3,7-diamino-10h-phenothiazine salts and their use

ABSTRACT

This invention pertains generally to the field of phenothiazine compounds, and more particularly to certain stably reduced phenothiazine compounds, specifically, certain 3,7 diamino-10H-phenothiazine (DAPTZ) compounds of the following formula wherein: each of R 1  and R 9  is independently selected from: —H; C 1-4 alkyl; C 2-4 alkenyl; and halogenated C 1-4 alkyl; each of R 3NA  and R 3NB  is independently selected from: —H; C 1-4 alkyl; C 2-4 alkenyl; and halogenated C 1-4 alkyl; each of R 7NA  and R 7NB  is independently selected from: —H; C 1-4 alkyl; C 2-4 alkenyl; and halogenated C 1-4 alkyl; each of HX 1  and HX 2  is independently a protic acid; and pharmaceutically acceptable salts, solvates, and hydrates thereof. These compounds are useful as drugs, for example, in the treatment of tauopathies, such as Alzheimer&#39;s disease, and also as prodrugs for the corresponding oxidized thioninium drugs (for example, methythioninium chloride, MTC).

RELATED APPLICATION

This application is related to U.S. patent application No. 60/786,690filed 29 Mar. 2006; the contents of which are incorporated herein byreference in their entirety.

TECHNICAL FIELD

This invention pertains generally to the field of phenothiazinecompounds, and more particularly to certain stably reduced phenothiazinecompounds, specifically, certain 3,7-diamino-10H-phenothiazine (DAPTZ)compounds, for example,N,N,N′,N′-tetramethyl-10H-phenothiazine-3,7-diamine bis(hydrogenchloride) and N,N,N′,N′-tetramethyl-10H-phenothiazine-3,7-diaminebis(hydrogen iodide). These compounds are useful as drugs, for example,in the treatment of tauopathies, such as Alzheimer's disease, and alsoas prodrugs for the corresponding oxidized thioninium drugs (forexample, methythioninium chloride, MTC).

BACKGROUND

A number of patents and publications are cited herein in order to morefully describe and disclose the invention and the state of the art towhich the invention pertains. Each of these references is incorporatedherein by reference in its entirety into the present disclosure, to thesame extent as if each individual reference was specifically andindividually indicated to be incorporated by reference.

Throughout this specification, including the claims which follow, unlessthe context requires otherwise, the word “comprise,” and variations suchas “comprises” and “comprising,” will be understood to imply theinclusion of a stated integer or step or group of integers or steps butnot the exclusion of any other integer or step or group of integers orsteps.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a pharmaceutical carrier” includes mixtures of two or moresuch carriers, and the like.

Ranges are often expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by the use of the antecedent “about,” itwill be understood that the particular value forms another embodiment.

Conditions of dementia are frequently characterised by a progressiveaccumulation of intracellular and/or extracellular deposits ofproteinaceous structures such as β-amyloid plaques and neurofibrillarytangles (NFTs) in the brains of affected patients. The appearance ofthese lesions largely correlates with pathological neurofibrillarydegeneration and brain atrophy, as well as with cognitive impairment(see, e.g., Mukaetova-Ladinska, E. B. et al., 2000, Am. J. Pathol., Vol.157, No. 2, pp. 623-636). Methythioninium chloride (MTC) and otherdiaminophenothiazines have been described as inhibitors of proteinaggregation in such diseases, that is, diseases in which proteinsaggregate pathologically (see, for example, WO 96/30766 and WO02/055720).

Methythioninium chloride (MTC) is currently used to treatmethemoglobinemia (a condition that occurs when the blood cannot deliveroxygen where it is needed in the body). MTC is also used as a medicaldye (for example, to stain certain parts of the body before or duringsurgery); a diagnostic (for example, as an indicator dye to detectcertain compounds present in urine); a mild urinary antiseptic; astimulant to mucous surfaces; a treatment and preventative for kidneystones; and in the diagnosis and treatment of melanoma.

MTC has been used to treat malaria either singly (see, e.g., Guttmann,P. and Ehrlich, P., 1891, “Uber die wirkung des methylenblau beimalaria,” Berl. Kiin. Woschenr., Vol. 28, pp. 953-956) or in combinationwith chloroquine (see, e.g., Schirmer, H., et al., 2003, “Methylene blueas an antimalarial agent,” Redox Report, Vol. 8, pp. 272-275;Rengelshausen, J., et al., 2004, “Pharmacokinetic interaction ofchloroquine and methylene blue combination against malaria,” EuropeanJournal of Clinical Pharmacology, Vol. 60, pp. 709-715). Malaria inhumans is caused by one of four protozoan species of the genusPlasmodium: P. falciparum, P. vivax, P. ovale, or P. malariae. Allspecies are transmitted by the bite of an infected female Anophelesmosquito. Occasionally, transmission occurs by blood transfusion, organtransplantation, needle-sharing, or congenitally from mother to fetus.Malaria causes 300-500 million infections worldwide and approximately 1million deaths annually. Drug resistance, however, is a major concernand is greatest for P. falciparum, the species that accounts for almostall malaria-related deaths. Drugs or drug combinations that arecurrently recommended for prophylaxis of malaria includechloroquine/proguanil hydrochloride, mefloquine, doxycycline, andprimaquine.

MTC (under the name Virostat®, from Bioenvision Inc., New York) hasshown potent viricidal activity in vitro. Specifically Virostat® iseffective against viruses such as HIV and West Nile Virus in laboratorytests. West Nile virus (WNV) is a potentially serious illness affectingthe central nervous system. The large majority of infected people willshow no visible symptoms or mild flu-like symptoms such as fever andheadache. About one in 150 will develop severe symptoms includingtremors, convulsions, muscle weakness, vision loss, numbness, paralysis,or coma. Generally, WNV is spread by the bite of an infected mosquito,but can also spread through blood transfusions, organ transplants,breastfeeding or during pregnancy from mother to child.

Virostat® is also currently in clinical trials for the treatment ofchronic Hepatitis C. Hepatitis C is a viral infection of the liver. Thevirus, HCV, is a major cause of acute hepatitis and chronic liverdisease, including cirrhosis and liver cancer. HCV is spread primarilyby direct contact with human blood. The major causes of HCV infectionworldwide are use of unscreened blood transfusions, and re-use ofneedles and syringes that have not been adequately sterilized. The WorldHealth Organization has declared hepatitis C a global health problem,with approximately 3% of the world's population infected with HCV and itvaries considerably by region. The prevalence in the US is estimated at1.3% or approximately 3.5 million people. Egypt has a population ofapproximately 62 million and contains the highest prevalence ofhepatitis C in the world, estimated at over 20% of the nation'sapproximately 62 million people.

MTC, when combined with light, can prevent the replication of nucleicacid (DNA or RNA). Plasma, platelets and red blood cells do not containnuclear DNA or RNA. When MTC is introduced into the blood components, itcrosses bacterial cell walls or viral membrane then moves into theinterior of the nucleic acid structure. When activated with light, thecompound then binds to the nucleic acid of the viral or bacterialpathogen, preventing replication of the DNA or RNA. Because MTC caninactivate pathogens, it has the potential to reduce the risk oftransmission of pathogens that would remain undetected by testing.

Oral and parenteral formulations of MTC are commercially available inthe United States, usually under the name Urolene Blue®.

SUMMARY OF THE INVENTION

One aspect of the invention pertains to certain compounds, specifically,certain 3,7-diamino-10H-phenothiazine (DAPTZ) compounds, as describedherein.

Another aspect of the invention pertains to a composition comprising aDAPTZ compound as described herein and a pharmaceutically acceptablecarrier or diluent.

Another aspect of the invention pertains to a pharmaceutical compositioncomprising a DAPTZ compound as described herein and a pharmaceuticallyacceptable carrier or diluent.

Another aspect of the invention pertains to a method of preparing apharmaceutical composition comprising admixing a DAPTZ compound asdescribed herein and a pharmaceutically acceptable carrier or diluent.

Another aspect of the present invention pertains to a method ofreversing and/or inhibiting the aggregation of a protein (e.g., a tauprotein, a synuclein, etc.), for example, aggregation of a proteinassociated with a neurodegenerative disease and/or clinical dementia,comprising contacting the protein with an effective amount of a DAPTZcompound, as described herein. Such a method may be performed in vitro,or in vivo.

Another aspect of the present invention pertains to a method oftreatment or prophylaxis of a disease condition in a subject comprisingadministering to said subject a prophylactically or therapeuticallyeffective amount of a DAPTZ compound, as described herein, preferably inthe form of a pharmaceutical composition.

Another aspect of the present invention pertains to a DAPTZ compound asdescribed herein for use in a method of treatment or prophylaxis (e.g.,of a disease condition) of the human or animal body by therapy.

Another aspect of the present invention pertains to use of a DAPTZcompound, as described herein, in the manufacture of a medicament foruse in the treatment or prophylaxis of a disease condition.

In one embodiment, the disease condition is a disease of proteinaggregation.

In one embodiment, the disease condition is a tauopathy, e.g., aneurodegenerative tauopathy, e.g., Alzheimer's disease.

In one embodiment, the disease condition is skin cancer, e.g., melanoma.

In one embodiment, the disease condition is a viral, bacterial orprotozoal disease condition, e.g., Hepatitis C, HIV, West Nile Virus(WNV), or malaria.

Another aspect of the present invention pertains to a method ofinactivating a pathogen in a sample (for example a blood or plasmasample), comprising the steps of introducing a DAPTZ compound, asdescribed herein, into the sample, and then exposing the sample tolight.

Another aspect of the present invention pertains to a kit comprising (a)a DAPTZ compound as described herein, preferably provided as apharmaceutical composition and in a suitable container and/or withsuitable packaging; and (b) instructions for use, for example, writteninstructions on how to administer the compound.

As will be appreciated by one of skill in the art, features andpreferred embodiments of one aspect of the invention will also pertainto other aspect of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of the percent reduced form (%) versus time (minutes)for each of three compounds, B1 (MTC), B3(N,N,N′,N′-Tetramethyl-10H-phenothiazine-3,7-diamine bis(hydrogenchloride)), and B6 (N,N,N′,N′-Tetramethyl-10H-phenothiazine-3,7-diaminebis(hydrogen iodide)), as determined using absorbance at 665 nm.

FIG. 2 is a graph of the percent reduced form (%) versus time (minutes)for each of three compounds, B1, B3, and B6, as determined usingabsorbance at 610 nm.

FIG. 3A shows the UV/visible absorption spectra for aqueous samples ofeach of three compounds, B1 (open circles, maximum at 605 nm), B3 (opensquares, maximum at 660 nm), and B6 (open triangles, maximum at 660 nm),after 20 minutes.

FIG. 3B shows the UV/visible absorption spectra for aqueous samples ofeach of three compounds, B1 (open circles, maximum at 605 nm), B3 (opensquares, maximum at 605 nm), and B6 (open triangles, maximum at 605 nm),after 3 hours.

FIG. 3C shows the UV/visible absorption spectra for aqueous samples ofeach of three compounds, B1 (open circles, maximum at 605 nm), B3 (opensquares, maximum at 605 nm), and B6 (open triangles, maximum at 605 nm),after 28 hours.

FIG. 4 shows the crystal structure ofN,N,N′,N′-tetramethyl-10H-phenothiazine-3,7-diamine bis(hydrogenbromide).

FIG. 5 shows the side-on view ofN,N,N′,N′-tetramethyl-10H-phenothiazine-3,7-diamine bis(hydrogenbromide).

FIG. 6 shows part of one helical column ofN,N,N′,N′-tetramethyl-10H-phenothiazine-3,7-diamine bis(hydrogenbromide) molecules in the crystal.

DETAILED DESCRIPTION OF THE INVENTION

Methythioninium Chloride (MTC) (also known as Methylene blue (MB);methylthionine chloride; tetramethylthionine chloride;3,7-bis(dimethylamino) phenothiazin-5-ium chloride; C.I. Basic Blue 9;tetramethylthionine chloride; 3,7-bis(dimethylamino) phenazathioniumchloride; Swiss blue; C.I. 52015; C.I. Solvent Blue 8; aniline violet;and Urolene Blue®) is a low molecular weight (319.86), water soluble,tricyclic organic compound of the following formula:

Methythioninium Chloride (MTC) (also known as Methylene Blue), perhapsthe most well known phenothiazine dye and redox indicator, has also beenused as an optical probe of biophysical systems, as an intercalator innanoporous materials, as a redox mediator, and in photoelectrochomicimaging.

MTC, a phenothiazin-5-ium salt, may conveniently be considered to be an“oxidized form” when considered in respect of the corresponding10H-phenothiazine compound,N,N,N′,N′-tetramethyl-10H-phenothiazine-3,7-diamine, which mayconveniently be considered to be a “reduced form”:

The “reduced form” (the “leuko form”) is known to be unstable, and isreadily and rapidly oxidized to give the corresponding “oxidized” form.

May et al. (Am J Physiol Cell Physiol, 2004, Vol. 286, pp. C1390-C1398)have shown that human erythrocytes sequentially reduce and take up MTC;that MTC itself is not taken up by the cells; that it is the reducedfrom of MTC that crosses the cell membrane; that the rate of uptake isenzyme dependent; and that both MTC and reduced MTC are concentrated incells (reduced MTC re-equilibrates once inside the cell to form MTC).

MTC and similar drugs are taken up in the gut and enter the bloodstream.Unabsorbed drug percolates down the alimentary canal, to the distal gut.One important undesired side-effect is the effect of the unabsorbed drugin the distal gut, for example, sensitisation of the distal gut and/orantimicrobial effects of the unabsorbed drug on flora in the distal gut,both leading to diarrhea. Therefore, it is desirable to minimize theamount of drug that percolates to the distal gut. By increasing thedrug's update in the gut (i.e., by increasing the drug'sbioavailability), dosage may be reduced, and the undesired side-effects,such as diarrhea, may be ameliorated.

Since it is the reduced form of MTC that is taken up by cells, it wouldbe desirable to administer the reduced form. This would also reducedreliance on the rate limiting step of enzymatic reduction.

The inventors have identified a class of compounds that may also beconsidered to be in the “reduced form” when considered in respect ofMTC, and which are surprisingly and unexpectedly stable. The compoundsmay therefore be described as “stabilized reduced forms,” for example,of MTC.

These compounds are themselves active as drugs, and may also serve asprodrugs, yielding, upon oxidation, the corresponding oxidized compounds(e.g., MTC), which are also active as drugs.

One representative member of this class of compounds is shown below.

Another representative member of this class of compounds is shown below.

The Compounds

In general, the present invention pertains certain3,7-diamino-10H-phenothiazine compounds of the following formula(collectively referred to herein as “diamino-phenothiazine compounds”and/or “DAPTZ compounds”):

wherein:

-   -   each of R¹ and R⁹ is independently selected from: —H, C₁₋₄alkyl,        C₂₋₄alkenyl, and halogenated C₁₋₄alkyl;    -   each of R^(3NA) and R^(3NB) is independently selected from: —H,        C₁₋₄alkyl, C₂₋₄alkenyl, and halogenated C₁₋₄alkyl;    -   each of R^(7NA) and R^(7NB) is independently selected from: —H,        C₁₋₄alkyl, C₂₋₄alkenyl, and halogenated C₁₋₄alkyl;    -   each of HX¹ and HX² is independently a protic acid;        and pharmaceutically acceptable salts, solvates, and hydrates        thereof.

Without wishing to be bound to any particular theory, the inventorsbelieve that it is possible, if not likely, that the compounds exist inthe following form:

Although the DAPTZ compounds are themselves salts, they may also beprovided in the form of a mixed salt (i.e., the DAPTZ in combinationwith another salt). Such mixed salts are intended to be encompassed bythe term “and pharmaceutically acceptable salts thereof”. Unlessotherwise specified, a reference to a particular compound also includessalts thereof.

The DAPTZ compounds may also be provided in the form of a solvate orhydrate. The term “solvate” is used herein in the conventional sense torefer to a complex of solute (e.g., compound, salt of compound) andsolvent. If the solvent is water, the solvate may be convenientlyreferred to as a hydrate, for example, a mono-hydrate, a di-hydrate, atri-hydrate, etc. Unless otherwise specified, a reference to aparticular compound also includes solvate forms thereof.

In one embodiment, the C₁₋₄alkyl groups are selected from: linearC₁₋₄alkyl groups, such as -Me, -Et, -nPr, -iPr, and -nBu; branchedC₃₋₄alkyl groups, such as -iPr, -iBu, -sBu, and -tBu; and cyclicC₃₋₄alkyl groups, such as -cPr and -cBu.

In one embodiment, the C₂₋₄alkenyl groups are selected from linearC₁₋₄alkenyl groups, such as —CH═CH₂ (vinyl) and —CH₂—CH═CH₂ (allyl).

In one embodiment, the halogenated C₁₋₄alkyl groups are selected from:—CF₃, —CH₂CF₃, and —CF₂CF₃.

The Groups R¹ and R⁹

In one embodiment, each of R¹ and R⁹ is independently —H, -Me, -Et, or—CF₃.

In one embodiment, each of R¹ and R⁹ is independently —H, -Me, or -Et.

In one embodiment, R¹ and R⁹ are the same.

In one embodiment, R¹ and R⁹ are different.

In one embodiment, each of R¹ and R⁹ is independently —H.

In one embodiment, each of R¹ and R⁹ is independently -Me.

In one embodiment, each of R¹ and R⁹ is independently -Et.

The Groups R^(3NA) and R^(3NB)

Each of R^(3NA) and R^(3NB) is independently selected from: —H,C₁₋₄alkyl, C₂₋₄alkenyl, and halogenated C₁₋₄alkyl.

In one embodiment, each of R^(3NA) and R^(3NB) is independently selectedfrom: C₁₋₄alkyl, C₂₋₄alkenyl, and halogenated C₁₋₄alkyl.

In one embodiment, each of R^(3NA) and R^(3NB) is independently -Me,-Et, -nPr, -nBu, —CH₂—CH═CH₂, or —CF₃.

In one embodiment, each of R^(3NA) and R^(3NB) is independently -Me,-nPr, -nBu, —CH₂—CH—CH₂, or —CF₃.

In one embodiment, each of R^(3NA) and R^(3NB) is independently -Me or-Et.

In one embodiment, R^(3NA) and R^(3NB) are the same.

In one embodiment, R^(3NA) and R^(3NB) are different.

In one embodiment, each of R^(3NA) and R^(3NB) is independently -Me.

In one embodiment, each of R^(3NA) and R^(3NB) is independently -Et.

The Groups R^(7NA) and R^(7NB)

Each of R^(7NA) and R^(7NB) is independently selected from: —H,C₁₋₄alkyl, C₂₋₄alkenyl, and halogenated C₁₋₄alkyl.

In one embodiment, each of R^(7NA) and R^(7NB) is independently selectedfrom: C₁₋₄alkyl, C₂₋₄alkenyl, and halogenated C₁₋₄alkyl.

In one embodiment, each of R^(7NA) and R^(7NB) is independently -Me,-Et, -nPr, -nBu, —CH₂—CH═CH₂, or —CF₃.

In one embodiment, each of R^(7NA) and R^(7NB) is independently -Me,-nPr, -nBu, —CH₂—CH═CH₂, or —CF₃.

In one embodiment, each of R^(7NA) and R^(7NB) is independently -Me or-Et.

In one embodiment, R^(7NA) and R^(7NB) are the same.

In one embodiment, R^(7NA) and R^(7NB) are different.

In one embodiment, each of R^(7NA) and R^(7NB) is independently -Me.

In one embodiment, each of R^(7NA) and R^(7NB) is independently -Et.

In one embodiment, R^(3NA) and R^(3NB) and R^(7NA) and R^(7NB) are thesame.

In one embodiment, R^(3NA) and R^(3NB) and R^(7NA) and R^(7NB) are asdefined herein, with the proviso that at least one of R^(3NA) andR^(3NB) and R^(7NA) and R^(7NB) is other than -Et.

Optional Provisos

In one embodiment, the compound is as defined herein, but with theproviso that: R^(3NA) and R^(3NB) and R^(7NA) and R^(7NB) are not each-Et.

In one embodiment, the compound is as defined herein, but with theproviso that:

if: each of R¹ and R⁹ is —H;then: R^(3NA) and R^(3NB) and R^(7NA) and R^(7NB) are not each -Et.

The Groups —N(R^(3NA))(R^(3NB)) and —N(R^(7NA))(R^(7NB))

In one embodiment:

each of R^(3NA) and R^(3NB) is independently C₁₋₄alkyl, C₂₋₄alkenyl, orhalogenated C₁₋₄alkyl;each of R^(7NA) and R^(7NB) is independently C₁₋₄alkyl, C₂₋₄alkenyl, orhalogenated C₁₋₄alkyl;optionally with the proviso that at least one of R^(3NA) and R^(3NB) andR^(7NA) and R^(7NB) is other than -Et.

In one embodiment:

each of R^(3NA) and R^(3NB) is independently -Me, -Et, -nPr, -nBu,—CH₂—CH═CH₂, or —CF₃;each of R^(7NA) and R^(7NB) is independently -Me, -Et, -nPr, -nBu,—CH₂—CH═CH₂, or —CF₃;optionally with the proviso that at least one of R^(3NA) and R^(3NB) andR^(7NA) and R^(7NB) is other than -Et.

In one embodiment:

each of R^(3NA) and R^(3NB) is independently -Me or -Et;each of R^(7NA) and R^(7NB) is independently -Me or -Et;optionally with the proviso that at least one of R^(3NA) and R^(3NB) andR^(7NA) and R^(7NB) is other than -Et.

In one embodiment, the groups —N(R^(3NA))(R^(3NB)) and—N(R^(7NA))(R^(7NB)) are the same.

In one embodiment, the groups —N(R^(3NA))(R^(3NB)) and—N(R^(7NA))(R^(7NB)) are different.

In one embodiment, each of the groups —N(R^(3NA))(R^(3NB)) and—N(R^(7NA))(R^(7NB)) is independently selected from: —NMe₂, —NEt₂,—N(nPr)₂, —N(Bu)₂, —NMeEt, —NMe(nPr), and —N(CH₂CH═CH₂)₂.

In one embodiment, the groups —N(R^(3NA))(R^(3NB)) and—N(R^(7NA))(R^(7NB)) are the same, and are independently selected from:—NMe₂, —NEt₂, —N(nPr)₂, —N(Bu)₂, —NMeEt, —NMe(nPr), and —N(CH₂CH═CH₂)₂.

In one embodiment, the groups —N(R^(3NA))(R^(3NB)) and—N(R^(7NA))(R^(7NB)) are the same, and are independently selected from:—NMe₂ and —NEt₂.

In one embodiment, each of the groups —N(R^(3NA))(R^(3NB)) and—N(R^(7NA))(R^(7NB)) is: —NMe₂ ⁺.

In one embodiment, at least one of the groups —N(R^(3NA))(R^(3NB)) and—N(R^(7NA))(R^(7NB)) is other than —NEt₂.

In one embodiment, each of the groups —N(R^(3NA))(R^(3NB)) and—N(R^(7NA))(R^(7NB)) is other than —NEt₂.

For example, in one embodiment, the groups —N(R^(3NA))(R^(3NB)) and—N(R^(7NA))(R^(7NB)) are the same, and are selected from: —NMe₂,—N(nPr)₂, —N(Bu)₂, —NMeEt, —NMe(nPr), and —N(CH₂CH═CH₂)₂.

The Groups HX¹ and HX²

Each of HX¹ and HX² is independently a protic acid.

Examples of protic acids include, for example, inorganic acids, such ashydrohalide acids (e.g., HCl, HBr, HI), nitric acid (HNO₃), sulphuricacid (H₂SO₄), and organic acids, such as carbonic acid (H₂CO₃) andacetic acid (CH₃COOH).

In one embodiment, each of HX¹ and HX² is independently a monoproticacid.

In one embodiment, each of HX¹ and HX² is independently a hydrohalideacid (i.e., a hydrohalic acid)

In one embodiment, each of HX¹ and HX² is independently selected fromHCl, HBr, and HI.

In one embodiment, HX¹ and HX² are the same.

In one embodiment, HX¹ and HX² are different.

In one embodiment, HX¹ and HX² are the same, and are independentlyselected from HCl, HBr, and HI. In this case, the compound (adiamino-phenothiazine compound) may conveniently be referred to as a“diamino-phenothiazine bis(hydrogen halide) salt”.

In one embodiment, HX¹ and HX² are each HCl. In this case, the compoundmay conveniently be referred to as a “diamino-phenothiazine bis(hydrogenchloride) salt”.

In one embodiment, HX¹ and HX² are each HBr. In this case, the compoundmay conveniently be referred to as a “diamino-phenothiazine bis(hydrogenbromide) salt”.

In one embodiment, HX¹ and HX² are each HI. In this case, the compoundmay conveniently be referred to as a “diamino-phenothiazine bis(hydrogeniodide) salt”.

Some Preferred Combinations

In one embodiment:

each of R¹ and R⁹ is independently —H, -Me, or -Et; andeach of the groups N(R^(3NA))(R^(3NB)) and —N(R^(7NA))(R^(7NB)) isindependently —NMe₂ or —NEt₂.

In one embodiment:

each of R¹ and R⁹ is independently —H, -Me, or -Et; andeach of the groups —N(R^(3NA))(R^(3NB)) and —N(R^(7NA))(R^(7NB)) isindependently —NMe₂.

In one embodiment:

each of R¹ and R⁹ is independently —H; andeach of the groups N(R^(3NA))(R^(3NB)) and —N(R^(7NA))(R^(7NB)) isindependently —NMe₂ or —NEt₂.

In one embodiment:

each of R¹ and R⁹ is independently —H; andeach of the groups —N(R^(3NA))(R^(3NB)) and —N(R^(7NA))(R^(7NB)) isindependently —NMe₂,

In one embodiment:

each of R¹ and R⁹ is independently —H, -Me, or -Et; andeach of the groups —N(R^(3NA))(R^(3NB)) and —N(R^(7NA))(R^(7NB)) isindependently —NMe₂ or —NEt₂; andeach of HX¹ and HX² is independently selected from HCl, HBr, and HI.

In one embodiment:

each of R¹ and R⁹ is independently —H, -Me, or -Et; andeach of the groups —N(R^(3NA))(R^(3NB)) and —N(R^(7NA))(R^(7NB)) isindependently —NMe₂; andeach of HX¹ and HX² is independently selected from HCl, HBr, and HI.

In one embodiment:

each of R¹ and R⁹ is independently —H; andeach of the groups —N(R^(3NA))(R^(3NB)) and —N(R^(7NA))(R^(7NB)) isindependently —NMe₂ or —NEt₂; andeach of HX¹ and HX² is independently selected from HCl, HBr, and HI.

In one embodiment:

each of R¹ and R⁹ is independently —H; andeach of the groups —N(R^(3NA))(R^(3NB)) and —N(R^(7NA))(R^(7NB)) isindependently —NMe₂; andeach of HX¹ and HX² is independently selected from HCl, HBr, and HI.

In one embodiment:

each of R¹ and R⁹ is independently —H; andeach of the groups —N(R^(3NA))(R^(3NB)) and —N(R^(7NA))(R^(7NB)) isindependently —NMe₂; andeach of HX¹ and HX² is HCl.

In one embodiment:

each of R¹ and R⁹ is independently —H; andeach of the groups —N(R^(3NA))(R^(3NB)) and —N(R^(7NA))(R^(7NB)) isindependently —NMe₂; andeach of HX¹ and HX² is HBr.

In one embodiment:

each of R¹ and R⁹ is independently —H; andeach of the groups —N(R^(3NA))(R^(3NB)) and —N(R^(7NA))(R^(7NB)) isindependently —NMe₂; andeach of HX¹ and HX² is HI.

Isotopic Variation

In one embodiment, one or more of the carbon atoms of the compound is¹¹C, ¹³C, or ¹⁴C.

In one embodiment, one or more of the carbon atoms of the compound is¹¹C.

In one embodiment, one or more of the carbon atoms of the compound is¹³C.

In one embodiment, one or more of the carbon atoms of the compound is¹⁴C.

In one embodiment, one or more of the nitrogen atoms of the compound is¹⁵N.

In one embodiment, one or more or all of the carbon atoms of one or moreor all of the groups R^(3NA), R^(3NB), R^(7NA), R^(7NB), R¹, R⁹, and R¹⁰is ¹¹C. (Or ¹³C.) (Or ¹⁴C.)

In one embodiment, one or more or all of the carbon atoms of one or moreor all of the groups R^(3NA), R^(3NB), R^(7NA), and R^(7NB) is ¹¹C. (Or¹³C.) (Or ¹⁴C.)

In one embodiment, the groups —N(R^(3NA))(R^(3NB)) and—N(R^(7NA))(R^(7NB)) are the same, and are: —N(¹¹CH₃)₂. (Or —N(¹³CH₃)₂.)(Or —N(¹⁴CH₃)₂.)

Compatible Combinations

All compatible combinations of the embodiments described above areexplicitly disclosed herein as if each combination was specifically andindividually recited.

SOME PREFERRED EMBODIMENTS

In one embodiment, the compound is selected from the followingcompounds, and pharmaceutically acceptable salts, solvates, and hydratesthereof.

In one embodiment, the compound is selected from the followingcompounds, and pharmaceutically acceptable salts, solvates, and hydratesthereof.

In one embodiment, the compound is selected from the followingcompounds, and pharmaceutically acceptable salts, solvates, and hydratesthereof.

Purity

The DAPTZ compounds of the present invention may conveniently bedescribed as being in a “stabilized reduced form”. The compounds oxidize(e.g., autoxidize) to give the corresponding oxidized forms. Thus, it islikely, if not inevitable, that compositions comprising the DAPTZcompounds of the present invention will contain, as an impurity, asleast some of the corresponding oxidized compound.

Thus, another aspect of the present invention pertains to DAPTZcompounds, as described herein, in substantially purified form and/or ina form substantially free from contaminants (e.g., the correspondingoxidized compound, other contaminants).

In one embodiment, the substantially purified form is at least 50% byweight pure, e.g., at least 60% by weight pure, e.g., at least 70% byweight pure, e.g., at least 80% by weight pure, e.g., at least 90% byweight pure, e.g., at least 95% by weight pure, e.g., at least 97% byweight pure, e.g., at least 98% by weight pure, e.g., at least 99% byweight pure.

In one embodiment, the contaminants represent no more than 50% byweight, e.g., no more than 40% by weight, e.g., no more than 30% byweight, e.g., no more than 20% by weight, e.g., no more than 10% byweight, e.g., no more than 5% by weight, e.g., no more than 3% byweight, e.g., no more than 2% by weight, e.g., no more than 1% byweight.

Product-by-Process

In one embodiment, the DAPTZ compound is one which is obtained by, or isobtainable by, a method as described herein.

Chemical Synthesis

Methods for the chemical synthesis of DAPTZ compounds of the presentinvention are described herein. These and/or other well known methodsmay be modified and/or adapted in known ways in order to facilitate thesynthesis of additional DAPTZ compounds within the scope of the presentinvention.

For example, a suitable phenothiazine may be converted to thecorresponding 3,7-dinitro-phenothiazine, for example, using sodiumnitrite with acetic acid and chloroform. The ring amino group may thenbe protected, for example, as the acetate, for example, using aceticanhydride and pyridine. The nitro groups may then be reduced to aminogroups, for example, using tin (II) chloride with ethanol. The aminogroups may then be substituted, for example, disubstituted, for example,methyl disubstituted, for example, using methyl iodide, sodiumhydroxide, DMSO, and tetra-n-butyl ammonium bromide. The amino group maythen be deprotected, for example, the N-acetyl group may be removed, forexample, using concentrated aqueous hydrochloride acid. Thecorresponding salt is then prepared, for example, using concentratedaqueous hydrochloric acid, for example, at the same time asdeprotection. An example of such a method is illustrated in thefollowing scheme.

Thus, another aspect of the invention pertains to a method of preparinga 3,7-diamino-10H-phenothiazine (DAPTZ) compound of the followingformula:

wherein R¹, R⁹, R^(3NA), R^(3NB), R^(7NA), R^(7NB), HX¹ and HX² are asdefined herein (for example, where HX¹ and HX² are each HCl), comprisingthe step of:

-   -   (vi) salt formation (SF).

In one embodiment, the method comprises the steps of:

-   -   (v) ring amino deprotection (DP); and    -   (vi) salt formation (SF).

In one embodiment, the method comprises the steps of:

-   -   (iv) amine substitution (AS),    -   optional (v) ring amino deprotection (DP), and    -   (vi) salt formation (SF).

In one embodiment, the method comprises the steps of

-   -   (iii) nitro reduction (NR),    -   (iv) amine substitution (AS),    -   (v) ring amino deprotection (DP), and    -   (vi) salt formation (SF).

In one embodiment, the method comprises the steps of

-   -   optional (ii) ring amino protection (AP),    -   (iii) nitro reduction (NR),    -   (iv) amine substitution (AS),    -   (v) ring amino deprotection (DP), and    -   (vi) salt formation (SF).

In one embodiment, the method comprises the steps of

-   -   (i) nitration (NO),    -   (ii) ring amino protection (AP),    -   (iii) nitro reduction (NR),    -   (iv) amine substitution (AS),    -   (v) ring amino deprotection (DP), and    -   (vi) salt formation (SF).

In one embodiment, the steps are performed in the order listed (i.e.,any step in the list is performed at the same time as, or subsequent to,the preceding step in the list).

In one embodiment, the step of (v) ring amino deprotection (DP) and thestep of (vi) salt formation (SF) are performed simultaneously (i.e., asone step).

In one embodiment, the nitration (NO) step is:

-   -   (i) nitration (NO), wherein a 10H-phenothiazine is converted to        a 3,7-dinitro-10H-phenothiazine, for example:

In one embodiment, nitration is performed using a nitrite, for example,sodium nitrite, for example, sodium nitrite with acetic acid andchloroform. In one embodiment, R¹⁰ is —H.

In one embodiment, the ring amino protection (AP) step is:

-   -   (ii) ring amino protection (AP), wherein the ring amino group        (—NH—) of a 3,7-dinitro-10H-phenothiazine is converted to a        protected ring amino group (—NR^(prot)), for example:

In one embodiment, ring amino protection is achieved as an acetate, forexample, using acetic anhydride, for example, using acetic anhydride andpyridine.

In one embodiment, the nitro reduction (NR) step is:

-   -   (iii) nitro reduction (NR), wherein each of the nitro (—NO₂)        groups of a protected 3,7-dinitro-10H-phenothiazine is converted        to an amino (—NH₂) group, for example:

In one embodiment, nitro reduction may be performed using, for example,tin (II) chloride, for example, tin (II) chloride with ethanol.

In one embodiment, the amine substitution (AS) step is:

-   -   (iv) amine substitution (AS), wherein each of the amino (—NH₂)        groups of a protected 3,7-diamino-10H-phenothiazine is converted        to disubstituted amino group, for example:

In one embodiment, amine substitution is performed using an alkylhalide, for example, an alkyl iodide, for example, methyl iodide, forexample, methyl iodide with sodium hydroxide, DMSO, and tetra-n-butylammonium bromide.

In one embodiment, the ring amino deprotection (DP) step is:

-   -   (v) ring amino deprotection (DP), wherein the protecting group,        R^(Prot), is removed, for example:

In one embodiment, ring amino deprotection may be performed using acid,for example, hydrochloric acid, for example, concentrated aqueoushydrochloric acid.

In one embodiment, the step is:

-   -   (vi) salt formation (SF), wherein the corresponding salt is        formed, for example:

In one embodiment, salt formation may be performed using acid, forexample, hydrochloric acid, for example, concentrated aqueoushydrochloric acid.

In one embodiment, the steps of ring amine deprotection and saltformation are performed simultaneously (i.e., as one step), for example,compound (1) is formed from compound (2) in one step.

In another approach, a suitable thioninium chloride (for example,methylthioninium chloride, MTC, also known as Methylene Blue) isconverted to the corresponding halide, for example, by reaction withpotassium iodide, for example, aqueous potassium iodide. The resultingthioninium iodide is then reduced, for example, with ethyl iodide andethanol, and the corresponding salt formed. A similar method isdescribed in Drew, H. D. K, and Head, F. S. H., “Derivatives ofMethylene-blue,” Journal of the Chemical Society, 1933, pp. 248-253. Anexample of such a method is illustrated in the following scheme.

Thus, another aspect of the invention pertains to a method of preparinga 3,7-diamino-10H-phenothiazine (DAPTZ) compound of the followingformula:

wherein R¹, R⁹, R^(3NA), R^(3NB), R^(7NA), R^(7NB), HX¹ and HX² are asdefined herein (for example, where HX¹ and HX² are each HI), comprisingthe step of:

-   -   (ii) reduction and iodide salt formation (RISF).

In one embodiment, the method comprises the steps of:

-   -   (i) iodide exchange (IE); and    -   (ii) reduction and iodide salt formation (RISF).

In one embodiment, the steps are performed in the order listed (i.e.,any step in the list is performed at the same time as, or subsequent to,the preceding step in the list).

In one embodiment, the iodide exchange (IE) step is:

-   -   (i) iodide exchange (IE), wherein a 3,7-di(disubstituted        amino)-thioninium salt is converted to the corresponding        3,7-di(disubstituted amino)-thioninium iodide, for example        (where Y⁻ is an anionic counter ion, for example, halide, for        example, chloride or bromide):

In one embodiment, iodide exchange (IE) is achieved by reaction withpotassium iodide, for example, aqueous potassium iodide.

In one embodiment, the reduction and iodide salt formation (RISF) stepis:

-   -   (ii) reduction and iodide salt formation (RISF), wherein a        3,7-di(disubstituted amino)-thioninium iodide is reduced and        converted to the corresponding 3,7-diamino-10H-phenothiazine        iodide compound, for example:

In one embodiment, reduction and iodide salt formation (RISF) isachieved by reaction with ethyl iodide, for example, ethyl iodide andethanol.

In another approach, an appropriate thioninium salt, for example, ethylthioninium semi zinc chloride, is simultaneously reduced and the ringamino group protected, for example, by reaction with phenylhydrazine,ethanol, acetic anhydride, and pyridine. The corresponding salt may thenbe prepared, for example, using concentrated aqueous hydrochloric acid,for example, at the same time as deprotection. An example of such amethod is illustrated in the following scheme.

Thus, another aspect of the invention pertains to a method of preparinga 3,7-diamino-10H-phenothiazine (DAPTZ) compound of the followingformula:

wherein R¹, R⁹, R^(3NA), R^(3NB), R⁷NA, R^(7NB), HX¹ and HX² are asdefined herein (for example, where HX¹ and HX² are each HI), comprisingthe step of:comprising the step of:

-   -   (iv) salt formation (SF).

In one embodiment, the method comprises the steps of

-   -   (iii) ring amino deprotection (DP), and    -   (iv) salt formation (SF).

In one embodiment, the method comprises the steps of

-   -   (ii) ring amino protection (AP),    -   (iii) ring amino deprotection (DP), and    -   (iv) salt formation (SF).

In one embodiment, the method comprises the steps of

-   -   (i) reduction (RED)    -   (ii) ring amino protection (AP),    -   (iii) ring amino deprotection (DP), and    -   (iv) salt formation (SF).

In one embodiment, the steps are performed in the order listed (i.e.,any step in the list is performed at the same time as, or subsequent to,the preceding step in the list).

In one embodiment, the step of (i) reduction (RED) and the step of (ii)ring amino protection (AP) are performed simultaneously (i.e., as onestep).

For example, in one embodiment, the combined reduction (RED) step andring amino protection (AP) step is:

-   -   (i) reduction (RED) and ring amino protection (AP), wherein a        3,7-di(disubstituted amino)-thioninium salt is reduced to give        the corresponding 3,7-di(disubstituted amino)-10H-phenothiazine,        and the ring amino group (—NH—) of the 3,7-di(disubstituted        amino)-10H-phenothiazine is converted to a protected ring amino        group (—R^(prot)) to give the corresponding protected        3,7-di(disubstituted amino)-10H-phenothiazine, for example:

In one embodiment, Y represents Cl⁻.

In one embodiment, the combined reduction (RED) step and ring aminoprotection (AP) step is achieved using phenylhydrazine and aceticanhydride, for example, phenylhydrazine, ethanol, acetic anhydride, andpyridine.

In one embodiment, the step of (iii) ring amino deprotection (DP) andthe step of (iv) salt formation (SF) are performed simultaneously (i.e.,as one step).

For example, in one embodiment, the combined ring amino deprotection(DP) step and salt formation (SF) step is:

-   -   (ii) ring amino deprotection (DP) and salt formation (SF),        wherein the protecting group of a protected 3,7-di(disubstituted        amino)-10H-phenothiazine is removed to give a        3,7-di(disubstituted amino)-10H-phenothiazine, and the        corresponding salt is formed, for example:

In one embodiment, the combined ring amino deprotection (DP) step andsalt formation (SF) step may be performed using acid, for example,hydrochloric acid, for example, concentrated aqueous hydrochloric acid.

In a similar approach, an appropriate thioninium chloride (e.g., methylthioninium chloride, ethyl thioninium chloride) is first reduced andacetylated to give the corresponding1-(3,7-bis-dimethylamino-phenothiazin-10-yl)-ethanone, for example, byreaction with hydrazine (NH₂NH₂), methyl hydrazine (MeNHNH₂), or sodiumborohydride (NaBH₄); and acetic anhydride ((H₃CCO)₂O); for example, inthe presence of a suitable base, for example, pyridine (C5H5N) orHunig's base (diisopropylethylamine, C₈H₁₉N), for example, in a suitablesolvent, for example, ethanol or acetonitrile. The reduced andacetylated compound is then deprotected (by removing the acetyl group),for example, by reaction with a suitable halic acid, for example,hydrochloric acid or hydrobromic acid, in a suitable solvent, forexample, ethanol, and optionally with the addition of a suitable ether,for example, diethyl ether.

Compositions

Another aspect of the invention pertains to a composition comprising aDAPTZ compound, as described herein, and a pharmaceutically acceptablecarrier or diluent.

Uses

Reversing and/or Inhibiting the Aggregation of a Protein

One aspect of the invention is the use of a DAPTZ compound, as describedherein, to regulate (e.g., to reverse and/or inhibit) the aggregation ofa protein, for example, aggregation of a protein associated with aneurodegenerative disease and/or clinical dementia. The aggregation maybe in vitro, or in vivo, and may be associated with a disease state asdiscussed below.

Thus, one aspect of the invention pertains to a method of regulating(e.g., reversing and/or inhibiting) the aggregation of a protein, forexample, aggregation of a protein associated with a neurodegenerativedisease and/or clinical dementia, comprising contacting the protein withan effective amount of a DAPTZ compound, as described herein. The methodmay be performed in vitro, or in vivo.

Similarly, one aspect of the invention pertains to a method ofregulating (e.g., reversing and/or inhibiting) the aggregation of aprotein in the brain of a mammal, which aggregation is associated with adisease state as described herein, the treatment comprising the step ofadministering to said mammal in need of said treatment, aprophylactically or therapeutically effective amount of a DAPTZcompound, as described herein, that is an inhibitor of said aggregation.

Methods of Treatment

Another aspect of the present invention pertains to a method oftreatment comprising administering to a patient in need of treatment aprophylactically or therapeutically effective amount of a DAPTZcompound, as described herein, preferably in the form of apharmaceutical composition.

Use in Methods of Therapy

Another aspect of the present invention pertains to a DAPTZ compound, asdescribed herein, for use in a method of treatment (e.g., of a diseasecondition) of the human or animal body by therapy.

Use in the Manufacture of Medicaments

Another aspect of the present invention pertains to use of a DAPTZcompound, as described herein, in the manufacture of a medicament foruse in treatment (e.g., of a disease condition).

In one embodiment, the medicament comprises the DAPTZ compound.

Disease Conditions Treated—Diseases of Protein Aggregation

The DAPTZ compounds of the present invention are useful in the treatmentor prophylaxis of diseases of protein aggregation.

Thus, in one embodiment, the disease condition is a disease of proteinaggregation, and, for example, the treatment is with an amount of aDAPTZ compound, as described herein, sufficient to inhibit theaggregation of the protein associated with said disease condition.

In general, the protein aggregation is that which arises from an inducedconformational polymerisation interaction, i.e., one in which aconformational change of the protein, or in a fragment thereof, givesrise to templated binding and aggregation of further (precursor) proteinmolecules in a self-propagating manner. Once nucleation is initiated, anaggregation cascade may ensue which involves the induced conformationalpolymerisation of further protein molecules, leading to the formation oftoxic product fragments in aggregates which are substantially resistantto further proteolysis. The protein aggregates thus formed are thoughtto be a proximal cause of disease states manifested asneurodegeneration, clinical dementia, and other pathological symptoms.

The following Table provides a listing of various disease-associatedaggregating proteins and the corresponding diseases of proteinaggregation.

Diseases of protein aggregation Aggregating Fibril domain and/or subunitProtein Disease mutations size (kDa) Reference Neuro-degenerativedisorders Prion protein Prion diseases Inherited and 27 Prusiner (1998)sporadic forms (CJD, nvCJD, Fatal PrP-27-30; many familial insomnia,mutations. Gerstmann-Straussler- Scheinker syndrome, Kuru) FibrillogenicGasset et al. domains: 113- (1992) 120, 178-191, 202-218. Tau proteinAlzheimer's disease, Inherited and 10-12 Wischik et al. Down's syndrome,sporadic forms (1988) FTDP-17, CBD, post- encephalitic parkinsonism,Pick's disease, parkinsonism with dementia complex of Guam Truncated tau(tubulin-binding domain) 297-391. Mutations in tau Hutton et al. inFTDP-17. (1998) Many mutations Czech et al. in presenilin (2000)proteins. Amyloid Alzheimer's disease, Inherited and 4 Glenner &β-protein Down's syndrome sporadic forms Wong, (1984) Amyloid β-protein; 1-42(3). 11 mutations in Goate et al. APP in rare (1991)families. Huntingtin Huntington's disease N-termini of 40 DiFiglia etal. protein with (1997) expanded glutamine repeats. AtaxinsSpinocerebellar ataxias Proteins with Paulson et al. (1, 2, 3, 7) (SCA1,2, 3, 7) expanded (1999) glutamine repeats. AtrophinDentarubropallidoluysian Proteins with Paulson et al. atrophy (DRPLA)expanded (1999) glutamine repeats. Androgen Spinal and bulbar Proteinswith Paulson et al. receptor muscular atrophy expanded (1999) glutaminerepeats. Neuroserpin Familial encephalopathy Neuroserpin; 57 Davis etal. with neuronal inclusion S49P, S52R. (1999) bodies (FENIB)α-Synuclein Parkinson's disease, Inherited and 19 Spillantini et al.dementia with Lewy sporadic forms (1998) bodies, multiple system atrophyA53T, A30P in Polymeropoulos rare autosomal- et al. (1997) dominant PDfamilies. Cystatin C Hereditary cerebral Cystatin C less 12-13Abrahamson et angiopathy (Icelandic) 10 residues; al. (1992) L68Q.Superoxide Amyotrophic lateral SOD1 mutations. Shibata et al. dismutase1 sclerosis (1996) Non-neuro-degenerative disorders Haemoglobin Sicklecell anaemia Haemoglobin Carrell & beta chain (S). Gooptu (1998)Inclusion body Many mutations. haemolysis Serpins α1-Antitrypsindeficiency Mutations Lomas et al. (emphysema, cirrhosis) (1992)Antithrombin deficiency Mutatons Carrell & (thromboembolic disease)Gooptu (1998) C1-inhibitor deficiency Mutations Carrell & (angioedema)Gooptu (1998) Immunoglobulin Plasma cell dyscrasias light chain or0.5-25  Westermark et light chain (primary systemic AL fragments. al.(1985) amyloidosis) Serum amyloid Reactive, secondary 76-residue 4.5-7.5Westermark et A systemic AA amyloidosis fragment (critical al. (1985)residues 2-12). Chronic inflammatory disease Transthyretin Familialamyloid Tetramer 10-14 Gustavsson et polyneuropathy dissociated to al.(1991) (systemic; FAP I) conformational monomer variant. Many mutations(some not associated with amyloid; several different types of disease).Senile cardiac Normal 10-14 Gustavsson et amyloidosis transthyretin al.(1991) Gelsolin Familial amyloidosis - D187Q leads to 9.5 Maury &Finnish type (FAP IV) truncated 173- Baumann 225/243 (critical (1990)residues 182- 192). β2- Haemodialysis β2-Microglobulin 12-25 Gorevic etal. Microglobulin amyloidosis (1985) Prostatic amyloid ApolipoproteinFamilial amyloid N-terminal 83-93 9 Booth et al. Al polyneuropathyresidues; G26R, (1997) (systemic; FAP III) W50R, L60R Lysozyme Familialvisceral Lysozyme or 14 Pepys et al. amyloidosis fragments (with (1993)or without I56T, D67H) Amylin (Islet Type II diabetes Fragments 3.9Westermark amyloid (NIDDM) (critical core of (1990) polypeptide) 20-29);no mutations Fibrinogen Hereditary renal Fibrinogen  7-10 Uemichi et al.α-chain amyloidosis fragments (1992) Procalcitonin Medullary carcinomaof Calcitonin 3.4 Sletten et al. thyroid fragments (1976) Atrialnatriuretic Cardiac amyloidosis ANF, no mutants 3.5 Johansson et factoral. (1987) Insulin Injection localised Insulin Dische et al. amyloidosis(1988) Other proteins (in vitro) Other proteins Chiti et al. formingamyloid (1999) References for the above Table: Abrahamson, M.,Jonsdottir, S., Olafsson, I. & Grubb, A. (1992) Hereditary cystatin Camyloid angiopathy identification of the disease-causing mutation andspecific diagnosis by polymerase chain reaction based analysis. 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As described in WO 02/055720 and U.S. patent application No. 60/786,700filed on 29 Mar. 2006 (title: Inhibitors of Protein Aggregation),diaminophenothiazines have utility in the inhibition of such proteinaggregating diseases.

Thus it will be appreciated that, except where context requiresotherwise, description of embodiments with respect to tau protein ortau-like proteins (e.g., MAP2), should be taken as applying equally tothe other proteins discussed herein (e.g., β-amyloid, synuclein, prion,etc.) or other proteins which may initiate or undergo a similarpathological aggregation by virtue of conformational change in a domaincritical for propagation of the aggregation, or which impartsproteolytic stability to the aggregate this formed (see, e.g., thearticle by Wischik et al. in “Neurobiology of Alzheimer's Disease”, 2ndEdition, 2000, Eds. Dawbarn, D. and Allen, S. J., The Molecular andCellular Neurobiology Series, Bios Scientific Publishers, Oxford). Allsuch proteins may be referred to herein as “aggregating diseaseproteins.”

Likewise, where mention is made herein of “tau-tau aggregation”, or thelike, this may also be taken to be applicable to other“aggregating-protein aggregation”, such as β-amyloid aggregation, prionaggregation, synuclein aggregation, etc. The same applies for “tauproteolytic degradation” etc.

Preferred Aggregating Disease Proteins

Preferred embodiments of the invention are based on tau protein. Theterm “tau protein,” as used herein, refers generally to any protein ofthe tau protein family. Tau proteins are characterised as being oneamong a larger number of protein families which co-purify withmicrotubules during repeated cycles of assembly and disassembly (see,e.g., Shelanski et al., 1973, Proc. Natl. Acad. Sci. USA, Vol. 70, pp.765-768), and are known as microtubule-associated-proteins (MAPs).Members of the tau family share the common features of having acharacteristic N-terminal segment, sequences of approximately 50 aminoacids inserted in the N-terminal segment, which are developmentallyregulated in the brain, a characteristic tandem repeat region consistingof 3 or 4 tandem repeats of 31-32 amino acids, and a C-terminal tail.

MAP2 is the predominant microtubule-associated protein in thesomatodendritic compartment (see, e.g., Matus, A., in “Microtubules”[Hyams and Lloyd, Eds.] pp. 155-166, John Wiley and Sons, New York,USA). MAP2 isoforms are almost identical to tau protein in the tandemrepeat region, but differ substantially both in the sequence and extentof the N-terminal domain (see, e.g., Kindler and Garner, 1994, Mol.Brain Res., Vol. 26, pp. 218-224). Nevertheless, aggregation in thetandem-repeat region is not selective for the tau repeat domain. Thus itwill be appreciated that any discussion herein in relation to tauprotein or tau-tau aggregation should be taken as relating also totau-MAP2 aggregation, MAP2-MAP2 aggregation, and so on.

In one embodiment, the protein is tau protein.

In one embodiment, the protein is a synuclein, e.g., α- or β-synuclein.

Where the protein is tau protein, in one embodiment of the presentinvention, there is provided a method of inhibiting production ofprotein aggregates (e.g. in the form of paired helical filaments (PHFs),optionally in neurofibrillary tangles (NFTs) in the brain of a mammal,the treatment being as described above.

Preferred Diseases of Protein Aggregation

Notably it is not only Alzheimer's disease (AD) in which tau protein(and aberrant function or processing thereof) may play a role. Thepathogenesis of neurodegenerative disorders such as Pick's disease andProgressive Supranuclear Palsy (PSP) appears to correlate with anaccumulation of pathological truncated tau aggregates in the dentategyrus and stellate pyramidal cells of the neocortex, respectively. Otherdementias include fronto-temporal dementia (FTD); parkinsonism linked tochromosome 17 (FTDP-17); disinhibition-dementia-parkinsonism-amyotrophycomplex (DDPAC); pallido-ponto-nigral degeneration (PPND); Guam-ALSsyndrome; pallido-nigro-luysian degeneration (PNLD); cortico-basaldegeneration (CBD) and others (see, e.g., the article by Wischik et al.in “Neurobiology of Alzheimer's Disease”, 2nd Edition, 2000, Eds.Dawbarn, D. and Allen, S. J., The Molecular and Cellular NeurobiologySeries, Bios Scientific Publishers, Oxford; especially Table 5.1). Allof these diseases, which are characterized primarily or partially byabnormal tau aggregation, are referred to herein as “tauopathies”.

Thus, in one embodiment, the disease condition is a tauopathy.

In one embodiment, the disease condition is a neurodegenerativetauopathy.

In one embodiment, the disease condition is Alzheimer's disease.

In one embodiment, treatment (e.g., treatment of a neurodegenerativetauopathy, e.g., Alzheimer's disease) may optionally be in combinationwith one or more other agents, for example, one or more cholinesteraseinhibitors (such as Donepezil (also known as Aricept™), Rivastigmine(also known as Exelon™), Galantamine (also known as Reminyl™), NMDAreceptor antagonists (such as Memantine (also known as Ebixa™,Namenda™), muscarinic receptor agonists, and/or inhibitors of amyloidprecursor protein processing that leads to enhanced generation ofbeta-amyloid.

Disease Conditions Treated—Other Disease Conditions

In one embodiment, the disease condition is skin cancer.

In one embodiment, the disease condition is melanoma.

In one embodiment, the disease condition is a viral, bacterial orprotozoal disease condition.

In one embodiment, the (protozoal) disease condition is malaria.

In this embodiment, treatment may be in combination with one or moreantimicrobial agents, for example, chloroquine and/or atovaquone.

In one embodiment, the (viral) disease condition is caused by HepatitisC, HIV, or West Nile Virus (WNV).

Other Uses

Another aspect of the present invention pertains to use of a DAPTZcompound, as described herein, in a method of inactivating a pathogen ina sample (for example a blood or plasma sample), comprising the steps ofintroducing the DAPTZ compound into the sample, and exposing the sampleto light.

For example, in one embodiment, the method comprises the steps ofintroducing the DAPTZ compound into the sample, and then exposing thesample to light.

Use as Ligands

The DAPTZ compounds that are capable of inhibiting the aggregation oftau protein will also be capable of acting as ligands or labels of tauprotein (or aggregated tau protein). Thus, in one embodiment, the DAPTZcompound is a ligand of tau protein (or aggregated tau protein).

Such DAPTZ compounds (ligands) may incorporate, be conjugated to, bechelated with, or otherwise be associated with, other chemical groups,such as stable and unstable detectable isotopes, radioisotopes,positron-emitting atoms, magnetic resonance labels, dyes, fluorescentmarkers, antigenic groups, therapeutic moieties, or any other moietythat may aid in a prognostic, diagnostic, or therapeutic application.

For example, in one embodiment, the DAPTZ compound is as defined herein,but with the additional limitation that the compound incorporates, isconjugated to, is chelated with, or is otherwise associated with, one ormore (e.g., 1, 2, 3, 4, etc.) detectable labels, for example, isotopes,radioisotopes, positron-emitting atoms, magnetic resonance labels, dyes,fluorescent markers, antigenic groups, or therapeutic moieties.

In one embodiment, the DAPTZ compound is a ligand as well as a label,e.g., a label for tau protein (or aggregated tau protein), andincorporates, is conjugated to, is chelated with, or is otherwiseassociated with, one or more (e.g., 1, 2, 3, 4, etc.) detectable labels.

For example, in one embodiment, the DAPTZ compound is as defined above,but with the additional limitation that the compound incorporates, isconjugated to, is chelated with, or is otherwise associated with, one ormore (e.g., 1, 2, 3, 4, etc.) detectable labels.

Labelled DAPTZ compounds (e.g., when ligated to tau protein oraggregated tau protein) may be visualised or detected by any suitablemeans, and the skilled person will appreciate that any suitabledetection means as is known in the art may be used.

For example, the DAPTZ compound (ligand-label) may be suitably detectedby incorporating a positron-emitting atom (e.g., ¹¹C) (e.g., as a carbonatom of one or more alkyl group substituents, e.g., methyl groupsubstituents) and detecting the compound using positron emissiontomography (PET) as is known in the art.

Such ¹¹C labelled DAPTZ compounds may be prepared by adapting themethods described herein in known ways, for example, in analogy to themethods described in WO 02/075318 (see FIGS. 11a, 11b, 12) and WO2005/030676.

Thus, another aspect of the present invention pertains to a method oflabelling tau protein (or aggregated tau protein) comprising the stepof: (i) contacting the tau protein (or aggregated tau protein) with aDAPTZ compound that incorporates, is conjugated to, is chelated with, oris otherwise associated with, one or more (e.g., 1, 2, 3, 4, etc.)detectable labels.

Another aspect of the present invention pertains to a method ofdetecting tau protein (or aggregated tau protein) comprising the stepsof: (i) contacting the tau protein (or aggregated tau protein) with aDAPTZ compound that incorporates, is conjugated to, is chelated with, oris otherwise associated with, one or more (e.g., 1, 2, 3, 4, etc.)detectable labels, and (ii) detecting the presence and/or amount of saidcompound bound to tau protein (or aggregated tau protein).

Another aspect of the present invention pertains to a method ofdiagnosis or prognosis of a tau proteinopathy in a subject believed tosuffer from the disease, comprising the steps of: (i) introducing intothe subject a DAPTZ compound capable of labelling tau protein oraggregated tau protein, particularly tau protein (e.g., a DAPTZ compoundthat incorporates, is conjugated to, is chelated with, or is otherwiseassociated with, one or more (e.g., 1, 2, 3, 4, etc.) detectablelabels); (ii) determining the presence and/or amount of said compoundbound to tau protein or aggregated tau protein in the brain of thesubject; and (iii) correlating the result of the determination made in(ii) with the disease state of the subject.

Another aspect of the present invention pertains to a DAPTZ compoundcapable of labelling tau protein or aggregated tau protein (e.g., aDAPTZ compound that incorporates, is conjugated to, is chelated with, oris otherwise associated with, one or more (e.g., 1, 2, 3, 4, etc.)detectable labels), for use in a method of diagnosis or prognosis of atau proteinopathy.

Another aspect of the present invention pertains to use of a DAPTZcompound capable of labelling tau protein or aggregated tau protein,particularly tau protein (e.g., a DAPTZ compound that incorporates, isconjugated to, is chelated with, or is otherwise associated with, one ormore (e.g., 1, 2, 3, 4, etc.) detectable labels), in a method ofmanufacture of a diagnostic or prognostic reagent for use in thediagnosis or prognosis of a tau proteinopathy.

Those skilled in the art will appreciate that instead of administeringDAPTZ ligands/labels directly, they could be administered in a precursorform, for conversion to the active form (e.g., ligating form, labellingform) by an activating agent present in, or administered to, the samesubject.

The ligands disclosed herein may be used as part of a method ofdiagnosis or prognosis. It may be used to select a patient fortreatment, or to assess the effectiveness of a treatment or atherapeutic (e.g., an inhibitor of tau protein aggregation) administeredto the subject.

Treatment

The term “treatment,” as used herein in the context of treating acondition, pertains generally to treatment and therapy, whether of ahuman or an animal (e.g., in veterinary applications), in which somedesired therapeutic effect is achieved, for example, the inhibition ofthe progress of the condition, and includes a reduction in the rate ofprogress, a halt in the rate of progress, regression of the condition,amelioration of the condition, and cure of the condition. Treatment as aprophylactic measure (i.e., prophylaxis, prevention) is also included.

The term “therapeutically-effective amount,” as used herein, pertains tothat amount of a DAPTZ compound, or a material, composition or dosagefrom comprising a DAPTZ compound, which is effective for producing somedesired therapeutic effect, commensurate with a reasonable benefit/riskratio, when administered in accordance with a desired treatment regimen.

Similarly, the term “prophylactically effective amount,” as used herein,pertains to that amount of a DAPTZ compound, or a material, compositionor dosage from comprising a DAPTZ compound, which is effective forproducing some desired prophylactic effect, commensurate with areasonable benefit/risk ratio, when administered in accordance with adesired treatment regimen.

The term “treatment” includes combination treatments and therapies, inwhich two or more treatments or therapies are combined, for example,sequentially or simultaneously. Examples of treatments and therapiesinclude, but are not limited to, chemotherapy (the administration ofactive agents, including, e.g., drugs, antibodies (e.g., as inimmunotherapy), prodrugs (e.g., as in photodynamic therapy, GDEPT,ADEPT, etc.); surgery; radiation therapy; and gene therapy.

For example, it may be beneficial to combine treatment with a DAPTZcompound as described herein with one or more other (e.g., 1, 2, 3, 4)agents or therapies.

The particular combination would be at the discretion of the physicianwho would select dosages using his/her common general knowledge anddosing regimens known to a skilled practitioner.

The agents (i.e., a DAPTZ compound as described here, plus one or moreother agents) may be administered simultaneously or sequentially, andmay be administered in individually varying dose schedules and viadifferent routes. For example, when administered sequentially, theagents can be administered at closely spaced intervals (e.g., over aperiod of 5-10 minutes) or at longer intervals (e.g., 1, 2, 3, 4 or morehours apart, or even longer periods apart where required), the precisedosage regimen being commensurate with the properties of the therapeuticagent(s).

The agents (i.e., a DAPTZ compound as described here, plus one or moreother agents) may be formulated together in a single dosage form, oralternatively, the individual agents may be formulated separately andpresented together in the form of a kit, optionally with instructionsfor their use.

Routes of Administration

The DAPTZ compound, or pharmaceutical composition comprising it, may beadministered to a subject/patient by any convenient route ofadministration, whether systemically/peripherally or topically (i.e., atthe site of desired action).

Routes of administration include, but are not limited to, oral (e.g., byingestion); buccal; sublingual; transdermal (including, e.g., by apatch, plaster, etc.); transmucosal (including, e.g., by a patch,plaster, etc.); intranasal (e.g., by nasal spray); ocular (e.g., byeyedrops); pulmonary (e.g., by inhalation or insufflation therapy using,e.g., via an aerosol, e.g., through the mouth or nose); rectal (e.g., bysuppository or enema); vaginal (e.g., by pessary); parenteral, forexample, by injection, including subcutaneous, intradermal,intramuscular, intravenous, intraarterial, intracardiac, intrathecal,intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal,intratracheal, subcuticular, intraarticular, subarachnoid, andintrasternal (including, e.g., intracatheter injection into the brain);by implant of a depot or reservoir, for example, subcutaneously orintramuscularly.

The Subject/Patient

The subject/patient may be an animal, a mammal, a placental mammal, arodent (e.g., a guinea pig, a hamster, a rat, a mouse), murine (e.g., amouse), a lagomorph (e.g., a rabbit), avian (e.g., a bird), canine(e.g., a dog), feline (e.g., a cat), equine (e.g., a horse), porcine(e.g., a pig), ovine (e.g., a sheep), bovine (e.g., a cow), a primate,simian (e.g., a monkey or ape), a monkey (e.g., marmoset, baboon), anape (e.g., gorilla, chimpanzee, orangutang, gibbon), or a human.

Furthermore, the subject/patient may be any of its forms of development,for example, a foetus.

In one preferred embodiment, the subject/patient is a human.

In one embodiment, the subject/patient is not a human.

Formulations

While it is possible for the DAPTZ compound to be used (e.g.,administered) alone, it is often preferable to present it as acomposition or formulation.

In one embodiment, the composition is a pharmaceutical composition(e.g., formulation, preparation, medicament) comprising a DAPTZcompound, as described herein, and a pharmaceutically acceptablecarrier, diluent, or excipient.

In one embodiment, the composition is a pharmaceutical compositioncomprising at least one DAPTZ compound, as described herein, togetherwith one or more other pharmaceutically acceptable ingredients wellknown to those skilled in the art, including, but not limited to,pharmaceutically acceptable carriers, diluents, excipients, adjuvants,fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers,solubilisers, surfactants (e.g., wetting agents), masking agents,colouring agents, flavouring agents, and sweetening agents.

In one embodiment, the composition further comprises other activeagents, for example, other therapeutic or prophylactic agents.

Suitable carriers, diluents, excipients, etc. can be found in standardpharmaceutical texts. See, for example, Handbook of PharmaceuticalAdditives, 2nd Edition (eds. M. Ash and 1. Ash), 2001 (SynapseInformation Resources, Inc., Endicott, N.Y., USA), Remington'sPharmaceutical Sciences, 20th edition, pub. Lippincott, Williams &Wilkins, 2000; and Handbook of Pharmaceutical Excipients, 2nd edition,1994.

Another aspect of the present invention pertains to methods of making apharmaceutical composition comprising admixing at least one[¹¹C]-radiolabelled DAPTZ compound, as defined herein, together with oneor more other pharmaceutically acceptable ingredients well known tothose skilled in the art, e.g., carriers, diluents, excipients, etc. Ifformulated as discrete units (e.g., tablets, etc.), each unit contains apredetermined amount (dosage) of the DAPTZ compound.

The term “pharmaceutically acceptable,” as used herein, pertains tocompounds, ingredients, materials, compositions, dosage forms, etc.,which are, within the scope of sound medical judgment, suitable for usein contact with the tissues of the subject in question (e.g., human)without excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio. Each carrier, diluent, excipient, etc. must also be “acceptable”in the sense of being compatible with the other ingredients of theformulation.

The formulations may be prepared by any methods well known in the art ofpharmacy. Such methods include the step of bringing into association theDAPTZ compound with a carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association the DAPTZ compound with carriers(e.g., liquid carriers, finely divided solid carrier, etc.), and thenshaping the product, if necessary.

The formulation may be prepared to provide for rapid or slow release;immediate, delayed, timed, or sustained release; or a combinationthereof.

Formulations suitable for parenteral administration (e.g., byinjection), include aqueous or non-aqueous, isotonic, pyrogen-free,sterile liquids (e.g., solutions, suspensions), in which the DAPTZcompound is dissolved, suspended, or otherwise provided (e.g., in aliposome or other microparticulate). Such liquids may additional containother pharmaceutically acceptable ingredients, such as anti-oxidants,buffers, preservatives, stabilisers, bacteriostats, suspending agents,thickening agents, and solutes which render the formulation isotonicwith the blood (or other relevant bodily fluid) of the intendedrecipient. Examples of excipients include, for example, water, alcohols,polyols, glycerol, vegetable oils, and the like. Examples of suitableisotonic carriers for use in such formulations include Sodium ChlorideInjection, Ringer's Solution, or Lactated Ringer's Injection. Typically,the concentration of the DAPTZ compound in the liquid is from about 1ng/ml to about 10 μg/ml, for example from about 10 ng/ml to about 1μg/ml. The formulations may be presented in unit-dose or multi-dosesealed containers, for example, ampoules and vials, and may be stored ina freeze-dried (lyophilised) condition requiring only the addition ofthe sterile liquid carrier, for example water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules, and tablets.

EXAMPLES OF SOME PREFERRED FORMULATIONS

One aspect of the present invention pertains to a dosage unit (e.g., apharmaceutical tablet or capsule) comprising 20 to 300 mg of a DAPTZcompound as described herein (e.g., obtained by, or obtainable by, amethod as described herein; having a purity as described herein; etc.),and a pharmaceutically acceptable carrier, diluent, or excipient.

In one embodiment, the dosage unit is a tablet.

In one embodiment, the dosage unit is a capsule.

In one embodiment, the amount is 30 to 200 mg.

In one embodiment, the amount is about 30 mg.

In one embodiment, the amount is about 60 mg.

In one embodiment, the amount is about 100 mg.

In one embodiment, the amount is about 150 mg.

In one embodiment, the amount is about 200 mg.

In one embodiment, the pharmaceutically acceptable carrier, diluent, orexcipient is or comprises one or both of a glyceride (e.g., Gelucire44/14®; lauroyl macrogol-32 glycerides PhEur, USP) and colloidal silicondioxide (e.g., 2% Aerosil 200®; Colliodal Silicon Dioxide PhEur, USP).

Dosage

It will be appreciated by one of skill in the art that appropriatedosages of the DAPTZ compound, and compositions comprising the DAPTZcompound, can vary from patient to patient. Determining the optimaldosage will generally involve the balancing of the level of therapeuticbenefit against any risk or deleterious side effects. The selecteddosage level will depend on a variety of factors including, but notlimited to, the activity of the particular compound, the route ofadministration, the time of administration, the rate of excretion of thecompound, the duration of the treatment, other drugs, compounds, and/ormaterials used in combination, the severity of the condition, and thespecies, sex, age, weight, condition, general health, and prior medicalhistory of the patient. The amount of compound and route ofadministration will ultimately be at the discretion of the physician,veterinarian, or clinician, although generally the dosage will beselected to achieve local concentrations at the site of action whichachieve the desired effect without causing substantial harmful ordeleterious side-effects.

Administration can be effected in one dose, continuously orintermittently (e.g., in divided doses at appropriate intervals)throughout the course of treatment. Methods of determining the mosteffective means and dosage of administration are well known to those ofskill in the art and will vary with the formulation used for therapy,the purpose of the therapy, the target cell(s) being treated, and thesubject being treated. Single or multiple administrations can be carriedout with the dose level and pattern being selected by the treatingphysician, veterinarian, or clinician.

In general, a suitable dose of the DAPTZ compound is in the range ofabout 100 ng to about 25 mg (more typically about 1 μg to about 10 mg)per kilogram body weight of the subject per day.

In one embodiment, the DAPTZ compound is administered to a human patientaccording to the following dosage regime: about 100 mg, 3 times daily.

In one embodiment, the DAPTZ compound is administered to a human patientaccording to the following dosage regime: about 150 mg, 2 times daily.

In one embodiment, the DAPTZ compound is administered to a human patientaccording to the following dosage regime: about 200 mg, 2 times daily.

EXAMPLES

The following examples are provided solely to illustrate the presentinvention and are not intended to limit the scope of the invention, asdescribed herein.

Chemical Synthesis Synthesis 1 3-Nitro-10H-phenothiazine

Sodium nitrite (20.00 g, 210 mmol) was added to a mixture of10H-phenothiazine (20.00 g, 50 mmol), chloroform (100 cm³), and aceticacid (20 cm³), and the mixture was stirred for 1 hour at roomtemperature. Acetic acid (20 cm³) was then added and the mixture wasstirred for a further 18 hours. The suspension was filtered and washedwith acetic acid, ethanol, water, and finally ethanol to give apurple/brown solid. The residue was dissolved in hot DMF and allowed tocool before filtering the di-nitro compound as a purple solid.Concentration of the DMF solution and washing the precipitate with waterand methanol gave the title mono-nitro compound (15 g, ˜50%) as a brownsolid; v_(max) (KBr)/cm⁻¹: 3328 (NH), 3278 (NH), 3229 (NH), 3119 (CH),3049 (CH), 1557 (NO₂), 1531 (NO₂); δ_(H) (250 MHz; DMSO): 6.64 (5H, m,ArH), 7.68 (1H, d, J 2.5, ArH), 7.79-7.84 (1H, dd, J 2.75, 6.5, ArH;δ_(C) (62.9 MHz; DMSO): 113.3 (ArC), 115.3 (ArC), 116.9 (ArC), 121.8(ArC), 123.6 (ArC), 123.7 (ArC), 124.6 (ArC), 126.4 (ArC), 128.1 (ArC),138.8 (ArC), 141.0 (ArC), 147.8 (ArC).

Synthesis 2 3,11-Dinitro-10H-phenothiazine

The procedure for the synthesis of 3-nitro-10H-phenothiazine wasfollowed using 3-nitro-10H-phenothiazine (10.00 g, 41 mmol), chloroform(40 cm³), acetic acid (2×10 cm³), and sodium nitrite (11.86 g, 173mmol). The residue obtained was recrystallised from DMF to yield thetitle di-nitro compound (6.60 g 56%) as purple needles; v_(max)(KBr)/cm⁻¹: 3331 (NH), 3294 (NH), 3229 (NH), 3101 (CH), 3067 (CH), 1602(NO₂), 1558 (NO₂); δ_(H) (250 MHz; DMSO): 6.73-6.76 (2H, d, J 9, ArH),7.78 (2H, s, ArH, 7.89-7.85 (2H, d, J 9, ArH).

Synthesis 3 1-(3,7-Dinitro-phenothiazin-10-yl)-ethanone

A solution of 3,11-dinitro-10H-phenothiazine (3.00 g, 10.37 mmol),acetic anhydride (15.88 g, 155.50 mmol), and pyridine (30 cm³) wasstirred at reflux for 18 hours. The warm solution was then carefullypoured over ice water. A precipitate formed and was filtered, dissolvedin dichloromethane, dried over magnesium sulphate, filtered, andconcentrated to give a brown/orange solid, which was purified by columnchromatography (SiO₂, ethyl acetate:petroleum ether, 2:3, loaded as adichloromethane solution) to give the title compound (2.46 g, 71%) as alight yellow solid which can be recrystallised from acetone to givelight yellow needles; v_(max) (KBr)/cm⁻¹: 3091 (CH), 3063 (CH), 1680(C═O), 1575 (NO₂), 1510 (NO₂); δ_(H) (250 MHz; CDCl₃): 2.28 (3H, s,CH₃), 7.65-7.69 (2H, d, J 9, ArH), 8.22-8.26 (2H, dd, J 2.75, 8.75,ArH), 8.33-8.32 (2H, d, J 2.5, ArH); δ_(C) (62.9 MHz; CDCl₃): 168.2(C═O), 146.3 (ArC), 143.3 (ArC), 133.6 (ArC), 127.8 (ArC), 123.4 (ArC),122.9 (ArC), 23.1 (CH₃); m/z (ES) 331.0 (80%, [M]⁺).

Synthesis 4 1-(3,7-Diamino-phenothiazin-10-yl)-ethanone

A mixture of 1-(3,7-dinitro-phenothiazin-10-yl)-ethanone (2 g, 6.04mmol), tin (II) chloride dihydrate (14.17 g, 62.8 mmol), and ethanol (50cm³) was heated to reflux and stirred at this temperature for 5 hours.The mixture was then cooled to room temperature and poured over icewater. The pH was adjusted to 7 with 5% sodium hydrogen carbonate beforethe product was extracted with ethyl acetate (3×50 cm³). The extractswere washed with brine and dried over magnesium sulphate, filtered, andconcentrated to give the title compound (1.64 g, 100%) as a purple bluesolid; v_(max)(KBr)/cm⁻¹: 3445 (NH), 3424 (NH), 3368 (NH), 3322 (NH),3203 (NH), 3054 (CH), 2995 (CH), 1706 (C═O), 1650 (NO₂), 1590 (NO₂);δ_(H) (250 MHz; CDCl₃): 2.01 (3H, s, CH₃), 5.09-5.43 (4H, brd s, NH),6.47-6.51 (2H, dd, J 1.5, 8.25, ArH), 6.61 (2H, s, ArH), 7.11-7.15 (2H,d, J 8, ArH); δ_(C) (62.9 MHz; CDCl₃): 169.1 (C═O), 147.2 (ArC), 128.1(ArC), 127.6 (ArC), 127.3 (ArC), 112.3 (ArC), 111.5 (ArC), 22.6 (CH₃);m/z (ES) 293.9 (95%, [M+H, Na]⁺), 272.0 (20%, [M+H]⁺), 227.9 (100%,[M+H, -Ac]⁺).

Synthesis 5 3,7-Diamino-phenothiazine bis(hydrogen chloride) (B4)

1-(3,7-Diamino-phenothiazin-10-yl)-ethanone (0.25 g, 0.921 mmol) wasdissolved in aqueous hydrochloric acid (5 N, 10 cm³) and the solutionwas heated to reflux and stirred for 30 minutes. Concentration of thereaction mixture gave the title compound as a light blue solid. δ_(H)(250 MHz; D₂O): 6.60 (2H, brd d, ArH), 7.07 (4H, brd s, ArH).

Synthesis 6 1-(3,7-Bis-dimethylamino-phenothiazin-10-yl)-ethanone

1-(3,7-Diamino-phenothiazin-10-yl)-ethanone (0.25 g 0.92 mmol) wasdissolved in DMSO (3 cm³). Toluene (10 cm³), iodomethane (1.96 g, 13.8mmol), tetrabutylammoniun bromide (50 mg), and finally aqueous sodiumhydroxide solution (50%, 1.25 cm³) were added. The mixture was stirredat room temperature for 2 hours. Additional aqueous sodium hydroxide(50%, 1.25 cm³) and iodomethane (1.96 g, 13.8 mmol) were then added. Themixture was allowed to stir for a further 3 hours at room temperaturebefore a third aliquot of aqueous sodium hydroxide (50%, 1.25 cm³) andiodomethane (1.96 g, 13.8 mmol) were added and the mixture stirred for afurther 18 hours. The thick suspension was washed with water (3×75 cm³)and the toluene extract collected. The water was extracted withdichloromethane (3×50 cm³) and the extracts combined with the toluene,and dried over magnesium sulphate, filtered, and concentrated to give adeep purple solid. The residue was purified by column chromatography(SiO₂, ethyl acetate:petroleum ether, 2:3, loaded as a dichloromethanesolution) to give the title compound product (0.12 g, 40%) as a lightpurple solid; v_(max)(KBr)/cm⁻¹: 2910 (CH), 2876 (CH), 2856 (CH), 2799(CH), 1659 (C═O), 1596 (NO₂), 1502 (NO₂); δ_(H) (250 MHz; CDCl₃): 2.16(3H, s, CH₃), 2.93 (12H, s, NCH₃), 6.59-6.62 (2H, d, J 8.5, ArH),6.69-6.71 (2H, d, J 2.75, ArH), 7.08-7.47 (2H, brd s, ArH); δ_(C) (62.9MHz; CDCl₃): 170.3 (C═O), 148.9 (ArC), 127.2 (ArC), 127.1 (ArC), 127.0(ArC), 110.9 (ArC), 110.7 (ArC), 40.7 (NCH₃), 22.9 (CH₃).

Synthesis 7 N,N,N′,N′-Tetramethyl-10H-phenothiazine-3,7-diaminebis(hydrogen chloride) (B3)

1-(3,7-Bis-dimethylamino-phenothiazin-10-yl)-ethanone (0.5 g, 1.84 mmol)was dissolved in aqueous hydrochloric acid (5 N, 15 cm³), and thesolution was heated to reflux temperature and stirred for 30 minutes.Concentration of the reaction mixture gave the title compound as agreen/blue solid; δ_(H) (250 MHz; D₂O): 3.18 (12H, s, NCH₃), 6.67 (2H,d, J8.5, ArH), 7.16 (4H, brd s, ArH); δ_(C) (62.9 MHz; D₂O): 144.3(ArC), 138.9 (ArC), 122.4 (ArC), 120.8 (ArC), 120.7 (ArC), 117.6 (ArC),48.9 (NCH₃).

Synthesis 8 Methylthioninium iodide

To a round bottom flask was added methylthioninium chloride (MTC,Methylene Blue) (2 g, 6.25 mmol) and water (50 cm³) and the mixturestirred for 10 minutes or until the solid dissolved. Potassium iodide(1.56 g, 9.4 mmol) was then added to the mixture and a green blacksuspension formed. The reaction was heated to boiling and allowed tocool naturally giving the title compound (2.03 g, 79%) as bright greenneedles. Anal. Calcd for C₁₆H₁₈N₃SI: C, 46.72; H, 4.41; N, 10.22; S,7.80; I, 30.85. Found: C, 46.30; H, 4.21; N, 10.14; S, 7.86; I, 29.34.

Synthesis 9 N,N,N′,N′-Tetramethyl-10H-phenothiazine-3,7-diaminebis(hydrogen iodide) (B6)

To a round bottom flask was added methylthioninium iodide (2 g, 4.86mmol), ethanol (100 cm³) and ethyl iodide (75.8 g, 486 mmol) and themixture was heated at reflux for 18 hours where the colour changed fromgreen/blue to brown with a yellow precipitate. Once cooled to roomtemperature, the mixture was filtered and washed with diethylether (20cm³) to give the title compound (1.99 g, 76%) as a light green solid.δ_(H) (250 MHz; D₂O): 3.20 (12H, s, NCH₃), 6.76 (2H, d, J 8.5, ArH),7.22 (2H, brd s, ArH); δ_(C) (62.9 MHz; D₂O): 145.0 (ArC), 139.3 (ArC),122.6 (ArC), 121.1 (ArC), 120.9 (ArC), 117.9 (ArC), 48.9 (NCH₃).

Synthesis 10 1-(3,7-Bis-diethylamino-phenothiazin-10-yl)-ethanone

To a dry 25 cm³ round bottom flask was added ethylthioninium zincchloride (0.5 g, 1.13 mmol) and ethanol (10 cm³). Phenylhydrazine (0.134g, 1.24 mmol) was then added dropwise under an atmosphere of nitrogen.The mixture was stirred 25° C. for 1 hour and concentrated under highvacuum. Pyridine (50 cm³) and acetic anhydride was added and the mixturestirred for 18 hours at 60° C. The solution was opened to ice/water (250cm³) and the organics were extracted into ethyl acetate (3×50 cm³). Theextracts were washed with saturated copper sulphate solution and driedover magnesium sulphate, filtered, and concentrated to give the crudeproduct as a brown oil, which was purified using flash columnchromatography with an eluent of 40% ethylacetate:60% petroleum spirit40-60° C. and silica 40-63μ 60 Å to give the title compound (0.18 g,41%) as a green glassy solid. δ_(H) (250 MHz; CDCl₃): 7.0-7.5 (2H, brds,ArH), 6.64 (2H, s, ArH), 6.52 (2H, d, ArH), 3.35 (8H, q, 7, NCH₂), 2.18(3H, s, CH₃), 1.16 (12H, t, 7, CH₃); δ_(C) (62.9 MHz; CDCl₃): 12.5(CH₃), 22.9 (CH₃), 44.6 (NCH₂), 110.1 (ArC), 127.4 (ArC), 146.5 (ArC),170.2 (C═O).

Synthesis 11 N,N,N′,N′-Tetraethyl-10H-phenothiazine-3,7-diaminebis(hydrogen chloride)

To a 25 cm³ round bottom flask was added3,7-diethylamino-10-acetyl-phenothiazine (0.125 g, 0.33 mmol) andaqueous hydrochloric acid (5 M, 5 cm³). The mixture was heated at 100°C. for 2 hours before cooling to room temperature and was concentratedto give the title compound (0.11 g, 81%) as a yellow green glassy solid.δ_(H) (250 MHz; CD₃OD): 7.07 (4H, brd, ArH), 6.65 (2H, brd, ArH), 3.35(8H, brd, NCH₂), 0.97 (12H, brd, CH₃); δ_(C) (62.9 MHz; CD₃OD): 10.8(CH₃), 55.1 (NCH₂), 116.6 (ArC), 120.4 (ArC), 121.5 (ArC), 123.6 (ArC),132.6 (ArC), 144.5 (ArC).

Synthesis 12 1-(3,7-Bis-dimethylamino-phenothiazin-10-yl)-ethanone

Synthesis using methylhydrazine/pyridine in two pots. To a 250 cm³ roundbottom flask placed under an atmosphere of argon was addedmethylthioninium chloride trihydrate (26.74 mmol, 10 g), ethanol (100cm³) and methylhydrazine (58.83 mmol, 2.71 g). The mixture was heated to40° C. and stirred for 2 hours. The yellow/green suspension was cooledto 5° C. and filtered under argon, washed with ethanol (20 cm³) anddried to give leuco-methylene blue as a light green solid. To the leucoproduct was added acetic anhydride (40 cm³) and pyridine (10 cm³) andthe solution was heated at 100° C. for 18 hours. The cooled mixture wasthen poured carefully over ice water while stirring to give aprecipitate, which was filtered, washed with water, and dried at 60° C.for 2 hours to yield the title compound (5.82 g, 66%) as a light brownsolid. Mp 137° C.; v_(max)(KBr)/cm⁻¹ 2910 (CH), 2876 (CH), 2856 (CH),2799 (CH), 1659 (C═O), 1596 (NO₂), 1502 (NO₂); δ_(H) (250 MHz; CDCl₃)2.16 (3H, s, CH₃), 2.93 (12H, s, NCH₃), 6.59-6.62 (2H, d, J 8.5, ArH),6.69-6.71 (2H, d, J 2.75, ArH), 7.08-7.47 (2H, brd s, ArH); δ_(C) (62.9MHz; CDCl₃) 170.3 (C═O), 148.9 (ArC), 127.2 (ArC), 127.1 (ArC), 127.0(ArC), 110.9 (ArC), 110.7 (ArC), 40.7 (NCH₃), 22.9 (CH₃); m/z (ES) 284.2(100%, [M-OAc]⁺), 328.1 (15%, [M+H]⁺), 350.1 (41%, [M+Na]⁺).

Synthesis 13 1-(3,7-Bis-dimethylamino-phenothiazin-10-yl)-ethanone

Synthesis using methylhydrazine/Hunig's base in one pot. To a 5000 cm³reactor vessel under an atmosphere of nitrogen was addedmethylthioninium chloride trihydrate (0.54 mol, 200 g) and acetonitrile(1000 cm³). Methylhydrazine (1.07 mol, 49.36 g) was added dropwise at1.5 mL per minute. The temperature of the mixture increased to 32° C.and was stirred for 20 minutes. The yellow/green suspension had aceticanhydride (5.35 mol, 541 g) added and then Hunig's base(diisopropylethylamine) (1.55 mol, 200 g) was added. The mixture washeated at 90° C. for 2 hours. The cooled mixture was then pouredcarefully into ice water (2000 cm³) in ten 200 cm³ portions whilestirring to give a precipitate. The precipitate was stirred for 45minutes before it was filtered, washed with water (3×250 cm³), and airdried for 30 minutes. The crude material was crystallised from hotethanol (2750 cm³) to yield the title compound (112.1 g, 64%) as a lightgrey solid. Mp 137° C.; v_(max)(KBr)/cm⁻¹ 2910 (CH), 2876 (CH), 2856(CH), 2799 (CH), 1659 (C═O), 1596 (NO₂), 1502 (NO₂); δ_(H) (250 MHz;CDCl₃) 2.16 (3H, s, CH₃), 2.93 (12H, s, NCH₃), 6.59-6.62 (2H, d, J 8.5,ArH), 6.69-6.71 (2H, d, J 2.75, ArH), 7.08-7.47 (2H, brd s, ArH); δ_(C)(62.9 MHz; CDCl₃) 170.3 (C═O), 148.9 (ArC), 127.2 (ArC), 127.1 (ArC),127.0 (ArC), 110.9 (ArC), 110.7 (ArC), 40.7 (NCH₃), 22.9 (CH₃); m/z (ES)284.2 (100%, [M-OAc]⁺), 328.1 (15%, [M+H]⁺), 350.1 (41%, [M+Na]⁺).

Synthesis 14 1-(3,7-Bis-dimethylamino-phenothiazin-10-yl)-ethanone

Synthesis using methylhydrazine/pyridine in one pot. To a 250 cm³ roundbottom flask under an atmosphere of nitrogen was added methylthioniniumchloride trihydrate (26.74 mmol, 10 g) and acetonitrile (50 cm³).Methylhydrazine (53.5 mmol, 2.46 g) was added in four equal portionsover a 30 minutes time period. The temperature of the mixture wasmaintained at 35° C. with a cold water bath and was stirred for 30minutes. The yellow/green suspension had acetic anhydride (267 mmol,27.3 g) and pyridine (80.2 mmol, 6.35 g) was added. The mixture washeated at 90° C. for 2 hours. The cooled mixture was then pouredcarefully into ice water (200 cm³) in ten equal portions while stirringto give a precipitate. The precipitate was stirred for 30 minutes beforeit was filtered, washed with water (3×50 cm³) and air dried for 30minutes. The crude material was crystallised from hot ethanol (120 cm³)to yield the title compound (5.97 g, 68%) as a light grey solid. Mp 137°C.; v_(max)(KBr)/cm⁻¹ 2910 (CH), 2876 (CH), 2856 (CH), 2799 (CH), 1659(C═O), 1596 (NO₂), 1502 (NO₂); δ_(H) (250 MHz; CDCl₃) 2.16 (3H, s, CH₃),2.93 (12H, s, NCH₃), 6.59-6.62 (2H, d, J 8.5, ArH), 6.69-6.71 (2H, d, J2.75, ArH), 7.08-7.47 (2H, brd s, ArH); δ_(C) (62.9 MHz; CDCl₃) 170.3(C═O), 148.9 (ArC), 127.2 (ArC), 127.1 (ArC), 127.0 (ArC), 110.9 (ArC),110.7 (ArC), 40.7 (NCH₃), 22.9 (CH₃); m/z (ES) 284.2 (100%, [M-OAc]⁺),328.1 (15%, [M+H]⁺), 350.1 (41%, [M+Na]⁺).

Synthesis 15 1-(3,7-Bis-dimethylamino-phenothiazin-10-yl)-ethanone

Synthesis using sodium borohydride/pyridine in one pot. To a 500 cm³round bottom flask under an atmosphere of nitrogen was addedmethylthioninium chloride trihydrate (0.134 mol, 50 g) and acetonitrile(250 cm³). Sodium borohydride (0.174 mol, 6.6 g) was added in four equalportions over a 30 minute time period. The temperature of the mixturewas maintained at 35° C. with a cold water bath and was stirred for 30minutes. The yellow/green suspension had acetic anhydride (0.535 mol, 55g) and pyridine (0.174 mol, 13.76 g) added. The mixture was heated at90° C. for 2 hours. The cooled mixture was then poured carefully intoice water (250 cm³) in ten equal portions while stirring to give aprecipitate. The precipitate was stirred for 30 minutes before it wasfiltered, washed with water (3×50 cm³), and air dried for 30 minutes.The crude material was crystallised from hot ethanol (500 cm³) to yieldthe title compound (26.7 g, 61%) as a light grey solid. Mp 137° C.;v_(max)(KBr)/cm⁻¹ 2910 (CH), 2876 (CH), 2856 (CH), 2799 (CH), 1659(C═O), 1596 (NO₂), 1502 (NO₂); δ_(H) (250 MHz; CDCl₃) 2.16 (3H, s, CH₃),2.93 (12H, s, NCH₃), 6.59-6.62 (2H, d, J 8.5, ArH), 6.69-6.71 (2H, d, J2.75, ArH), 7.08-7.47 (2H, brd s, ArH); δ_(c) (62.9 MHz; CDCl₃) 170.3(C═O), 148.9 (ArC), 127.2 (ArC), 127.1 (ArC), 127.0 (ArC), 110.9 (ArC),110.7 (ArC), 40.7 (NCH₃), 22.9 (CH₃); m/z (ES) 284.2 (100%, [M-OAc]⁺),328.1 (15%, [M+H]⁺), 350.1 (41%, [M+Na]⁺).

Synthesis 16 1-(3,7-Bis-dimethylamino-phenothiazin-10-yl)-ethanone

Synthesis using sodium borohydride/Hunig's base in one pot. To a 500 cm³round bottom flask under an atmosphere of nitrogen was addedmethylthioninium chloride trihydrate (80.2 mmol, 30 g) and acetonitrile(150 cm³). Sodium borohydride (104 mmol, 3.94 g) was added in four equalportions over a 30 minute time period. The temperature of the mixturewas maintained at 35° C. with a cold water bath and was stirred for 30minutes. The yellow/green suspension had acetic anhydride (321 mmol,32.75 g) and Hunig's base (diisopropylethylamine) (120 mmol, 15.55 g)added. The mixture was heated at 90° C. for 2 hours. The cooled mixturewas then poured carefully into ice water (200 cm³) in ten equal portionswhile stirring to give a precipitate. The precipitate was stirred for 30minutes before it was filtered, washed with water (3×50 cm³), and airdried for 30 minutes. The crude material was crystallised from hotethanol (300 cm³) to yield the title compound (13.55 g, 52%) as a lightgrey solid. Mp 137° C.; v_(max)(KBr)/cm⁻¹ 2910 (CH), 2876 (CH), 2856(CH), 2799 (CH), 1659 (C═O), 1596 (NO₂), 1502 (NO₂); δ_(H) (250 MHz;CDCl₃) 2.16 (3H, s, CH₃), 2.93 (12H, s, NCH₃), 6.59-6.62 (2H, d, J 8.5,ArH), 6.69-6.71 (2H, d, J2.75, ArH), 7.08-7.47 (2H, brd s, ArH); δ_(C)(62.9 MHz; CDCl₃) 170.3 (C═O), 148.9 (ArC), 127.2 (ArC), 127.1 (ArC),127.0 (ArC), 110.9 (ArC), 110.7 (ArC), 40.7 (NCH₃), 22.9 (CH₃); m/z (ES)284.2 (100%, [M-OAc]⁺), 328.1 (15%, [M+H]⁺), 350.1 (41%, [M+Na]⁺).

Synthesis 17 1-(3,7-Bis-dimethylamino-phenothiazin-10-yl)-ethanone

Synthesis using hydrazine monohydrate/pyridine in one pot. To a 250 cm³round bottom flask under an atmosphere of nitrogen was addedmethylthioninium chloride trihydrate (26.74 mmol, 10 g) and acetonitrile(50 cm³). Hydrazine monohydrate (58.8 mmol, 2.95 g) was added and themixture was heated to reflux and stirred for 10 minutes before coolingto 25° C. The yellow/green suspension had acetic anhydride (424 mmol,43.3 g) and pyridine (124 mmol, 9.78 g) added. The mixture was heated at90° C. for 2 hours. The cooled mixture was then poured carefully intoice water (100 cm³) in ten equal portions while stirring to give aprecipitate. The precipitate was stirred for 30 minutes before it wasfiltered, washed with water (3×50 cm³), and air dried for 30 minutes.The crude material was crystallised from hot ethanol (100 cm³) to yieldthe title compound (4.87 g, 56%) as a light grey solid. Mp 137° C.;v_(max)(KBr)/cm⁻¹ 2910 (CH), 2876 (CH), 2856 (CH), 2799 (CH), 1659(C═O), 1596 (NO₂), 1502 (NO₂); δ_(H) (250 MHz; CDCl₃) 2.16 (3H, s, CH₃),2.93 (12H, s, NCH₃), 6.59-6.62 (2H, d, J8.5, ArH), 6.69-6.71 (2H, d, J2.75, ArH), 7.08-7.47 (2H, brd s, ArH); δ_(C) (62.9 MHz; CDCl₃) 170.3(C═O), 148.9 (ArC), 127.2 (ArC), 127.1 (ArC), 127.0 (ArC), 110.9 (ArC),110.7 (ArC), 40.7 (NCH₃), 22.9 (CH₃); m/z (ES) 284.2 (100%, [M-OAc]⁺),328.1 (15%, [M+H]⁺), 350.1 (41%, [M+Na]⁺).

Synthesis 18 1-(3,7-Bis-dimethylamino-phenothiazin-10-yl)-ethanone

Synthesis using hydrazine monohydrate/Hunig's base in one pot. To a 250cm³ round bottom flask under an atmosphere of nitrogen was addedmethylthioninium chloride trihydrate (80.2 mmol, 30 g) and acetonitrile(150 cm³). Hydrazine monohydrate (176.5 mmol, 8.84 g) was added and themixture was heated to reflux and stirred for 10 minutes before coolingto 25° C. The yellow/green suspension had acetic anhydride (794 mmol,81.2 g) and Hunig's base (diisopropylethylamine) (232 mmol, 29.97 g)added. The mixture was heated at 90° C. for 2 hours. The cooled mixturewas then poured carefully into ice water (400 cm³) in ten equal portionswhile stirring to give a precipitate. The precipitate was stirred for 30minutes before it was filtered, washed with water (3×100 cm³), and airdried for 30 minutes. The crude material was crystallised from hotethanol (400 cm³) to yield the title compound (17.15 g, 65%) as a lightgrey solid. Mp 137° C.; v_(max)(KBr)/cm⁻¹ 2910 (CH), 2876 (CH), 2856(CH), 2799 (CH), 1659 (C═O), 1596 (NO₂), 1502 (NO₂); δ_(H) (250 MHz;CDCl₃) 2.16 (3H, s, CH₃), 2.93 (12H, s, NCH₃), 6.59-6.62 (2H, d, J 8.5,ArH), 6.69-6.71 (2H, d, J 2.75, ArH), 7.08-7.47 (2H, brd s, ArH); δ_(C)(62.9 MHz; CDCl₃) 170.3 (C═O), 148.9 (ArC), 127.2 (ArC), 127.1 (ArC),127.0 (ArC), 110.9 (ArC), 110.7 (ArC), 40.7 (NCH₃), 22.9 (CH₃); m/z (ES)284.2 (100%, [M-OAc]⁺), 328.1 (15%, [M+H]⁺), 350.1 (41%, [M+Na]⁺).

Synthesis 19 3,11-Dinitro-10H-phenothiazine

10H-Phenothiazine (20.00 g, 100 mmol), dichloromethane (100 cm³) andacetic acid (40 cm³) had sodium nitrite (20.07 g, 300 mmol) added andthe mixture was stirred for 10 minutes at room temperature. Additionalacetic acid (40 cm³), dichloromethane (100 cm³) and sodium nitrite(20.07 g, 300 mmol) were then added. A further 120 cm³ of acetic acidwas added to try and break up the thick reaction mixture. The mixturewas stirred for 3 hours. The suspension was filtered and washed with 100cm³ each of ethanol, water, and finally ethanol to give a purple/brownsolid. The residue was stirred in hot DMF and allowed to cool beforefiltering the dinitro product, which was washed with ethanol (150 cm³)and dried to give the title compound (24.88 g, 86%) as a brown solid;v_(max)(KBr)/cm⁻¹ 3331 (NH), 3294 (NH), 3229 (NH), 3101 (CH), 3067 (CH),1602 (NO₂), 1558 (NO₂); δ_(H) (250 MHz; DMSO) 6.73-6.76 (2H, d, J 9,ArH), 7.78 (2H, s, Arm), 7.89-7.85 (2H, d, J 9, ArH).

Synthesis 20 1-(3,7-Bis-diethylamino-phenothiazin-10-yl)-ethanone

To a 250 cm³ round bottom flask under an atmosphere of nitrogen wasadded ethylthioninium nitrate monohydrate (7.13 mmol, 3 g) andacetonitrile (20 cm³). Hydrazine monohydrate (16.4 mmol, 0.82 g) wasadded and the mixture was heated to reflux and stirred for 10 minutesbefore cooling to 25° C. The brown solution had acetic anhydride (114mmol, 11.65 g) and Hunig's base (diisopropylethylamine) (21.4 mmol, 2.77g) was added. The mixture was heated at 90° C. for 2 hours. The cooledmixture was then poured carefully into ice water (40 cm³) in ten equalportions while stirring to give a precipitate. The precipitate wasstirred for 30 minutes before it was filtered, washed with water (3×25cm³) and air dried for 30 minutes. The crude material was crystallisedfrom hot ethanol (50 cm³) to yield the title compound (1.73 g, 63%) as alight grey solid. δ_(H) (250 MHz; CDCl₃) 7.0-7.5 (2H, brds, ArH), 6.64(2H, s, ArH), 6.52 (2H, d, ArH), 3.35 (8H, q, 7, NCH₂), 2.18 (3H, s,CH₃), 1.16 (12H, t, 7, CH₃); δ_(C) (62.9 MHz; CDCl₃) 12.5 (CH₃), 22.9(CH₃), 44.6 (NCH₂), 110.1 (ArC), 127.4 (ArC), 146.5 (ArC), 170.2 (C═O).

Synthesis 21 N,N,N′,N′-Tetraethyl-10H-phenothiazine-3,7-diaminebis(hydrogen chloride)

To a round bottom flask was added1-(3,7-bis-diethylamino-phenothiazin-10-yl)-ethanone (0.5 g, 1.30 mmol),ethanol (5 cm³), and hydrochloric acid (37%, 1.3 cm³) and the solutionwas heated at 80° C. for 1 hour. Once cooled to room temperature, themixture was concentrated giving the title compound (0.54 g, 100%) as alight green glass. δ_(H) (250 MHz; CD₃OD) 7.07 (4H, brd, ArH), 6.65 (2H,brd, ArH), 3.35 (8H, brd, NCH₂), 0.97 (12H, brd, CH₃); δ_(C) (62.9 MHz;CD₃OD) 10.8 (CH₃), 55.1 (NCH₂), 116.6 (ArC), 120.4 (ArC), 121.5 (ArC),123.6 (ArC), 132.6 (ArC), 144.5 (ArC).

Synthesis 22 N,N,N′,N′-Tetraethyl-10H-phenothiazine-3,7-diaminebis(hydrogen bromide)

To a round bottom flask was added1-(3,7-bis-diethylamino-phenothiazin-10-yl)-ethanone (0.5 g, 1.30 mmol),ethanol (5 cm³), and hydrobromic acid (48%, 0.75 cm³) and the solutionwas heated at 80° C. for 1 hour. Once cooled to room temperature, themixture was concentrated giving the title compound (0.65 g, 100%) as alight yellow glass. δ_(H) (250 MHz; D₂O) 7.05 (4H, brd, ArH), 6.79 (2H,brd d, ArH), 3.43 (8H, brd, NCH₂), 1.05 (12H, brd t, CH₃); δ_(C) (62.9MHz; D₂O) 12.3 (CH₃), 56.2 (NCH₂), 117.9 (ArC), 121.4 (ArC), 122.4(ArC), 124.5 (ArC), 133.5 (ArC), 145.1 (ArC).

Synthesis 23 N,N,N′,N′-Tetramethyl-10H-phenothiazine-3,7-diaminebis(hydrogen chloride)

To a round bottom flask was added1-(3,7-bis-dimethylamino-phenothiazin-10-yl)-ethanone (1 g, 3.05 mmol),ethanol (10 cm³), and hydrochloric acid (37%, 3 cm³) and the solutionwas heated at 80° C. for 1 hour. Once cooled to room temperature,diethyl ether was added while stirring until a constant turbid solutionwas obtained. After some time, a precipitate formed, which was filteredand washed with diethyl ether (10 cm³) giving the title compound (0.98g, 90%) as a light green solid. Mp (dec) 230° C.; v_(max)(KBr)/cm⁻¹3500-3229 (NH), 3061 (CH), 3021 (CH), 2948 (CH), 2879 (CH), 2679 (CH),2601 (CH), 1604 (CH), 1483 (CH), 1318 (CH); δ_(H) (250 MHz; D₂O) 3.18(12H, s, NCH₃), 6.67 (2H, d, J 8.5, ArH), 7.16 (4H, brd s, ArH; δ_(C)(62.9 MHz; D₂O) 144.3 (ArC), 138.9 (ArC), 122.4 (ArC), 120.8 (ArC),120.7 (ArC), 117.6 (ArC), 48.9 (NCH₃); m/z (ES) 286.1 (100%, [M−H,2Cl]⁺), 285.1 (40%), 284.1 (41%, [M-3H, 2Cl]⁺).

Synthesis 24 N,N,N′,N′-Tetramethyl-10H-phenothiazine-3,7-diaminebis(hydrogen bromide)

To a round bottom flask was added1-(3,7-bis-dimethylamino-phenothiazin-10-yl)-ethanone (1 g, 3.05 mmol),ethanol (10 cm³), and hydrobromic acid (48%, 4 cm³) and the solution washeated at 80° C. for 1 hour. Once cooled to room temperature, aprecipitate formed, which was filtered and washed with diethyl ether (10cm³) giving the product (1.22 g, 89%) as a light mustard solid. Mp (dec)230° C.; v_(max)(KBr)/cm⁻¹ 3500-3229 (NH), 3061 (CH), 3021 (CH), 2948(CH), 2879 (CH), 2679 (CH), 2601 (CH), 1604 (CH), 1483 (CH), 1318 (CH);δ_(H) (250 MHz; D₂O) 3.18 (12H, s, NCH₃), 6.66 (2H, d, J 8.75, ArH),7.15 (4H, s, ArH); δ_(C) (62.9 MHz; D₂O) 144.3 (ArC), 138.9 (ArC), 122.4(ArC), 120.8 (ArC), 120.7 (ArC), 117.6 (ArC), 48.9 (NCH₃).

Synthesis 25 N,N,N′,N′-Tetraethyl-10H-phenothiazine-3,7-diaminebis(hydrogen bromide)

To a round bottom flask was added1-(3,7-bis-diethylamino-phenothiazin-10-yl)-ethanone (1.0 g, 2.60 mmol),methanol (10 cm³), and hydrobromic acid (48%, 2.94 cm³) and the solutionwas heated at 80° C. for 1 hour. Once cooled to 5° C., the mixture haddiethyl ether added, giving a cloudy solution. The solution was stirredfor 30 minutes and gave the title compound (0.83 g, 63%) as a lightyellow solid. δ_(H) (250 MHz; D₂O) 7.05 (4H, brd, ArH), 6.79 (2H, brd d,ArH), 3.43 (8H, brd, NCH₂), 1.05 (12H, brd t, CH₃); δ_(C) (62.9 MHz;D₂O) 12.3 (CH₃), 56.2 (NCH₂), 117.9 (ArC), 121.4 (ArC), 122.4 (ArC),124.5 (ArC), 133.5 (ArC), 145.1 (ArC).

Stability Studies

The DAPTZ compounds of the present invention are stably reduced (i.e.,are in a stably reduced form). For example, they are stable in the solidform, for example, for at least 1 week, e.g., at least 2 weeks, e.g., atleast 1 month, e.g., at least 2 months, e.g., at least 1 year (e.g., atroom temperature, e.g., 18-25° C., e.g., in a sealed container).

One sample of compound B3(N,N,N′,N′-Tetramethyl-10H-phenothiazine-3,7-diamine bis(hydrogenchloride)), in solid form, was found to be substantially reduced evenafter 2 years in storage.

Once the DAPTZ compounds are dissolved in water (i.e., in the form of anaqueous solution), they slowly oxidize (giving the solution a bluecolour), typically over a period of 1 to 3 hours.

The stability of two DAPTZ compounds of the present invention wasstudied, specifically, B3(N,N,N′,N′-Tetramethyl-10H-phenothiazine-3,7-diamine bis(hydrogenchloride)), and B6 (N,N,N′,N′-Tetramethyl-10H-phenothiazine-3,7-diaminebis(hydrogen iodide)). MTC was used as a standard.

The compounds were weighed into universal containers. Enough water wasadded to yield a 1 mM solution, and the mixture stirred to dissolve thesolid. The absorbance was determined at 610 nm and 665 nm for 50 μLsamples (in triplicate) of each of the solutions at various time points.The initial time point taken was at 10 minutes as the compounds tooktime to dissolve completely. A UV/visible spectrum was also recorded attime points 20 minutes, 3 hours, and 18 hours.

The percent reduced form (%) was calculated assuming that the 10 minutereading for MTC represented 0% reduced, and that a blank represented100% reduced (colourless).

FIG. 1 is a graph of the percent reduced form (%) versus time (minutes)for each of three compounds, B1, B3, and B6, as determined usingabsorbance at 665 nm.

FIG. 2 is a graph of the percent reduced form (%) versus time (minutes)for each of three compounds, B1, B3, and B6, as determined usingabsorbance at 610 nm.

FIGS. 3A, 3B, and 3C show the UV/visible absorption spectra for aqueoussamples of each of three compounds, B1 (open circles), B3 (opensquares), and B6 (open triangles), after 20 minutes (FIG. 3A), 3 hours(FIG. 3B), and 18 hours (FIG. 3C).

These data demonstrate that the DAPTZ compounds (stabilized reducedforms) remain substantially stable (>50%) for at least 1 hour, and thatcompound B6 remains substantially stable (>50%) for almost 3 hours.However, after about 18 hours, the compounds are not significantlydifferent from MTC. See, for example, FIG. 3C, where the spectra arealmost indistinguishable.

Additionally, the rate of autoxidation was found to be slower for the“iodide” compound (Compound B6) as compared to the “chloride” compound(Compound B3), suggesting that the rate of autoxidation depends upon thecounterion. Although the difference in rate was small, it may besignificant in drug formulation. Other salts may be more stable againstoxidation.

A batch of N,N,N′,N′-tetramethyl-10H-phenothiazine-3,7-diaminebis(hydrogen bromide) was prepared in April 2006 and analysed by NMR.After 10 months of storage in the dark at room temperature, the solidmaterial was analysed once more and the NMR data was found to beidentical. The colour of the solid remained consistent over time. Itappears that the molecule, in this form, is stable under theseconditions over this time period.

Biological Studies

Methods: In vitro Assay for Establishing B50

These methods are described in detail in WO 96/30766. Briefly, afragment of tau corresponding to the core repeat domain, which has beenadsorbed to a solid phase substrate, is able to capture solublefull-length tau and bind tau with high affinity. This associationconfers stability against proteolytic digestion of the aggregated taumolecules. The process is self-propagating, and can be blockedselectively by prototype pharmaceutical agents.

More specifically, truncated tau (residues 297-390; dGA) diluted incarbonate buffer (pH 9.6) was bound to the assay plate, and full-lengthtau (T40) was added in the aqueous phase. The aqueous phase bindingbuffer contained 0.05% Tween-20 and 1% gelatine in phosphate-bufferedsaline (pH 7.4). Bound tau was detected using mAb 499 that recognises anN-terminal epitope within the aqueous phase full-length tau but thatfails to recognise the solid phase-bound truncated tau fragment.

The concentration of compound required to inhibit the tau-tau binding by50% is referred to as the B50 value.

Methods: Cell-Based Assay for Establishing EC50

These methods are described in more detail in WO 02/055720. Briefly,fibroblast cells (3T6) express full-length tau (“T40”) under control ofan inducible promoter, and low constitutive levels of the PHF-core taufragment (12 kD fragment). When T40 expression is induced, it undergoesaggregation-dependent truncation within the cell, N-terminally at ˜αα295 and C-terminally at ˜αα 390, thereby producing higher levels of the12 kD PHF-core domain fragment. Production of the 12 kD fragment can beblocked in a dose-dependent manner by tau-aggregation inhibitors.Indeed, the quantitation of inhibitory activity of compounds withrespect to proteolytic generation of the 12 kD fragment within cells canbe described entirely in terms of the same parameters that describeinhibition of tau-tau binding in vitro. That is, the extent ofproteolytic generation of the 12 kD fragment within cells is determinedentirely by the extent of tau-tau binding through the repeat domain. Theavailability of the relevant proteases within the cell is non-limiting.

Results are expressed as the concentration at which there is a 50%inhibition of generation of the 12 kD fragment. This is referred to asthe EC50 value.

Methods: Toxicity in Cells (LD50) and Therapeutic Index (Rxl)

Toxicity of the compounds described herein was assessed in the cellbased assay used to assess EC50. Toxicity was measured by cell numbersdetermined after 24 hours exposure to the compound using a lactatedehydrogenase assay kit TOX-7 (Sigma Biosciences) according to themanufacturer's instructions after lysis of remaining cells.Alternatively, a kit from Promega UK (CytoTox 96) was used, againaccording to the manufacturer's instructions.

The therapeutic index (Rxl) was calculated as: Rxl=LD50/EC50.

The data are summarised in the following Table.

Biological Data Compound B50 EC50 LD50 RxI B3 57.3 0.50 44.0 88 B4 23.52.23 — — B6 494 0.38 115 303 MTC 218 0.59 65.0 110 B3:N,N,N′,N′-Tetramethyl-10H-phenothiazine-3,7-diamine bis(hydrogenchloride). B4: 10H-phenothiazine-3,7-diamine bis(hydrogen chloride). B6:N,N,N′,N′-Tetramethyl-10H-phenothiazine-3,7-diamine bis(hydrogeniodide). MTC: Methylthioninium chloride.

Partition Coefficient Studies

It is well known that the partition coefficient for a drug in an organicphase/water system (typically an n-octanol/water system), usuallyreported as the logarithm (i.e., log₁₀P), is a good indicator for thebiological activity of that drug. See, e.g., Hansch, C., et al., 1964,J. Am. Chem. Soc., Vol. 86, pp. 1616-1626; Kubinyi, H., 1977, J. Med.Chem., Vol. 20, pp. 625-629. It is believed that this is because theabsorption of a compound depends on its partition between the biologicalmembrane and the aqueous phase. Partition coefficients are also usefulin separation techniques and in the prediction of the solubility ofdrugs.

In the context of drug-like substances, hydrophobicity is related toabsorption, bioavailability, hydrophobic drug-receptor interactions,metabolism, and toxicity. Low hydrophilicities, and therefore highlog₁₀P values, may cause poor absorption or permeation. It has beenshown for compounds to have a reasonable probability of being wellabsorbed, their log₁₀P value must not be greater than 5.0. Thedistribution of calculated log₁₀P values of more than 3000 drugs on themarket underlines this fact.

Many methods for determining partition coefficients are known. In thisstudy, aqueous solutions of selected compounds were shaken withn-octanol and aliquots concentrations in each phase determined byvisible spectrophotometry. The log₁₀P was then calculated for eachcompound, using the following formula:

log₁₀P=log₁₀[Drug]_(octanol)−log₁₀[Drug]_(water)=log₁₀([Drug]_(octanol)/[Drug]_(water))

The data are summarised in the following table. The DAPTZ compounds ofthe present invention were found to have log₁₀P values expected fordrug-like molecules.

Partition Coefficient Data λ_(max) Absorbance for Absorbance forCompound (octanol/water) n-octanol phase water phase Log₁₀P MTC 665/6600.217 4.83 −1.35 B3 664/660 0.083 0.111 −0.13 B6 658.4/662.4 0.179 0.563−0.498 B3: N,N,N′,N′-Tetramethyl-10H-phenothiazine-3,7-diaminebis(hydrogen chloride). B6:N,N,N′,N′-Tetramethyl-10H-phenothiazine-3,7-diamine bis(hydrogeniodide). MTC: Methylthioninium chloride.

Crystal Structure

FIG. 4 shows the crystal structure ofN,N,N′,N′-tetramethyl-10H-phenothiazine-3,7-diamine bis(hydrogenbromide). The crystal structure shows three crystallographicallydistinct bromide ions. Br1 and Br2 occupy special positions with 2-foldsymmetry, whereas the organic main molecule and Br3 occupy generalpositions. Hence, the overall stoichiometry is C₁₆H₂₁N₃SBr₂.

FIG. 5 shows the side-on view ofN,N,N′,N′-tetramethyl-10H-phenothiazine-3,7-diamine bis(hydrogenbromide) and reveals the non-planarity; the dihedral angle between theouter benzene rings is 11.0 (3) degrees.

FIG. 6 shows part of one helical column ofN,N,N′,N′-tetramethyl-10H-phenothiazine-3,7-diamine bis(hydrogenbromide) molecules in the crystal.

1. A compound selected from compounds of the following formula andpharmaceutically acceptable salts, solvates, and hydrates thereof:

wherein: each of R¹ and R⁹ is independently selected from: —H,C₁₋₄alkyl, C₂₋₄alkenyl, and halogenated C₁₋₄alkyl; each of R^(3NA) andR^(3NB) is independently selected from: —H, C₁₋₄alkyl, C₂₋₄alkenyl, andhalogenated C₁₋₄alkyl; each of R^(7NA) and R^(7NB) is independentlyselected from: —H, C₁₋₄alkyl, C₂₋₄alkenyl, and halogenated C₁₋₄alkyl;each of HX¹ and HX² is independently a protic acid.
 2. A compoundaccording to claim 1, wherein each of R¹ and R⁹ is independently —H,-Me, -Et, or —CF₃.
 3. A compound according to claim 1, wherein each ofR¹ and R⁹ is independently —H, -Me, or -Et.
 4. A compound according toclaim 1, wherein R¹ and R⁹ are the same.
 5. A compound according toclaim 1, wherein each of R¹ and R⁹ is independently —H.
 6. A compoundaccording to claim 1, wherein each of R^(3NA) and R^(3NB) isindependently -Me, -Et, -nPr, -nBu, —CH₂—CH═CH₂, or —CF₃.
 7. A compoundaccording to claim 1, wherein R^(3NA) and R^(3NB) are the same.
 8. Acompound according to claim 1, wherein each of R^(7NA) and R^(7NB) isindependently -Me, -Et, -nPr, -nBu, —CH₂—CH═CH₂, or —CF₃.
 9. A compoundaccording to claim 1, wherein R^(7NA) and R^(7NB) are the same.
 10. Acompound according to claim 1, with the proviso that: at least one ofR^(3NA) and R^(3NB) and R^(7NA) and R^(7NB) is other than -Et.
 11. Acompound according to claim 1, with the proviso that: if: each of R¹ andR⁹ is —H; then: R^(3NA) and R^(3NB) and R^(7NA) and R^(7NB) are not each-Et.
 12. A compound according to claim 1, wherein each of the groups—N(R^(3NA))(R^(3NB)) and —N(R^(7NA))(R^(7NB)) is independently selectedfrom: —NMe₂, —NEt₂, —N(nPr)₂, —N(Bu)₂, —NMeEt, —NMe(nPr), and—N(CH₂CH═CH₂)₂.
 13. A compound according to claim 1, wherein the groups—N(R^(3NA))(R^(3NB)) and —N(R^(7NA))(R^(7NB)) are the same, and areselected from: —NMe₂ and —NEt₂.
 14. A compound according to claim 1,wherein the groups —N(R^(3NA))(R^(3NB)) and —N(R^(7NA))(R^(7NB)) are thesame.
 15. A compound according to claim 1, with the proviso that: eachof the groups —N(R^(3NA))(R^(3NB)) and —N(R^(7NA))(R^(7NB)) is otherthan —NEt₂.
 16. A compound according to claim 1, wherein the groups—N(R^(3NA))(R^(3NB)) and —N(R⁷NA)(R^(7NB)) are the same, and areselected from: —NMe₂, —N(nPr)₂, —N(Bu)₂, —NMeEt, —NMe(nPr), and—N(CH₂CH═CH₂)₂.
 17. A compound according to claim 1, wherein each of thegroups —N(R^(3NA))(R^(3NB)) and —N(R^(7NA))(R^(7NB)) is: —NMe₂.
 18. Acompound according to claim 1, wherein each of HX¹ and HX² isindependently a monoprotic acid.
 19. A compound according to claim 1,wherein each of HX¹ and HX² is independently a hydrohalide acid.
 20. Acompound according to claim 1, wherein each of HX¹ and HX² isindependently selected from HCl, HBr, and HI.
 21. A compound accordingto claim 1, wherein HX¹ and HX² are each HCl.
 22. A compound accordingto claim 1, wherein HX¹ and HX² are each HBr.
 23. (canceled)
 24. Acompound according to claim 1, selected from compounds of the followingformula, and pharmaceutically acceptable salts, solvates, and hydratesthereof:


25. A compound according to claim 1, selected from compounds of thefollowing formula, and pharmaceutically acceptable salts, solvates, andhydrates thereof:


26. (canceled)
 27. A compound according to claim 1, in substantiallypurified form.
 28. A compound according to claim 1, wherein one or moreof the carbon atoms of the compound is ¹¹C.
 29. (canceled)
 30. Apharmaceutical composition comprising a compound according to claim 1,and a pharmaceutically acceptable carrier or diluent.
 31. A method ofpreparing a pharmaceutical composition comprising admixing a compoundaccording to claim 1, and a pharmaceutically acceptable carrier ordiluent. 32-49. (canceled)
 50. A method of treatment or prophylaxis of adisease of protein aggregation in a subject comprising administering tosaid subject a prophylactically or therapeutically effective amount of acompound according to claim
 1. 51. A method of treatment or prophylaxisof a tauopathy in a subject comprising administering to said subject aprophylactically or therapeutically effective amount of a compoundaccording to claim
 1. 52. A method of treatment or prophylaxis of aneurodegenerative tauopathy in a subject comprising administering tosaid subject a prophylactically or therapeutically effective amount of acompound according to claim
 1. 53. A method of treatment or prophylaxisof Alzheimer's disease in a subject comprising administering to saidsubject a prophylactically or therapeutically effective amount of acompound according to claim
 1. 54. A method of treatment or prophylaxisof skin cancer or melanoma in a subject comprising administering to saidsubject a prophylactically or therapeutically effective amount of acompound according to claim
 1. 55. A method of treatment or prophylaxisof a viral, bacterial or protozoal disease condition in a subjectcomprising administering to said subject a prophylactically ortherapeutically effective amount of a compound according to claim
 1. 56.A method of treatment or prophylaxis of Hepatitis C, HIV, or West NileVirus (WNV) in a subject comprising administering to said subject aprophylactically or therapeutically effective amount of a compoundaccording to claim
 1. 57. A method of treatment or prophylaxis ofmalaria in a subject comprising administering to said subject aprophylactically or therapeutically effective amount of a compoundaccording to claim
 1. 58. A method of reversing and/or inhibiting theaggregation of a protein comprising contacting the protein with aneffective amount of a compound according to claim
 1. 59. A method ofreversing and/or inhibiting the aggregation of a protein in the brain ofa mammal, which aggregation is associated with a disease state, themethod comprising the step of administering to said mammal in need ofsaid treatment, a prophylactically or therapeutically effective amountof a compound according to claim
 1. 60. A method of inactivating apathogen in a sample, comprising the steps of introducing a compoundaccording to claim 1 into the sample, and then exposing the sample tolight.
 61. A pharmaceutical composition comprising a compound of thefollowing formula:

and a pharmaceutically acceptable carrier or diluent.
 62. A method oftreatment of a neurodegenerative tauopathy in a subject comprisingadministering to said subject a therapeutically effective amount of acomposition according to claim
 61. 63. A method of treatment ofAlzheimer's disease in a subject comprising administering to saidsubject a therapeutically effective amount of a composition according toclaim
 61. 64. A pharmaceutical composition comprising a compound of thefollowing formula:

and a pharmaceutically acceptable carrier or diluent.
 65. A method oftreatment of a neurodegenerative tauopathy in a subject comprisingadministering to said subject a therapeutically effective amount of acomposition according to claim
 64. 66. A method of treatment ofAlzheimer's disease in a subject comprising administering to saidsubject a therapeutically effective amount of a composition according toclaim 64.